Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin

Adrian Allen1 and Gunnar Flemström2

1Physiological Sciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom; and 2Division of Physiology, Department of Neuroscience, Uppsala University, Uppsala, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum.

acid-base transporters; cystic fibrosis transmembrane conductance regulator channel; surface pH gradient; mucus gels; trefoil peptides


MORE THAN A CENTURY AGO in 1892, Schierbeck (205) demonstrated high values of PCO2 in dog stomach, and in 1898, Pavlov (180) postulated that an alkaline mucus layer protects the gastric mucosal surface. Hollander (109), some 50 years later, proposed a two-component protective gastric mucosal barrier of an alkaline mucus layer and a rapidly regenerating underlying epithelium. This two-component hypothesis emphasized an alkaline secretion of nonparietal origin in the intact stomach, but intraluminal neutralization could not at that time be experimentally distinguished from back diffusion of secreted acid into the epithelium. Metabolism-dependent secretion of bicarbonate (HCO3) by intact gastric mucosa was first demonstrated in the mid-1970s by Flemström and coworkers (61, 66, 75) and was subsequently shown in the duodenum (64, 67, 214). A stable, unstirred layer, provided by surface mucus, was presumed to be a prerequisite for maintaining a pH higher at the epithelial surface than that in an acidic luminal solution (79, 244). The presence of a firm mucus gel layer adherent to gastric and duodenal mucosa was first directly demonstrated in the early 1980s by Allen and coworkers through visualization of unfixed mucosal sections and rheological studies (7, 15, 31, 131). At the same time, dynamic proof of the existence of a gastroduodenal mucus bicarbonate barrier to luminal acid came from demonstrations of a pH gradient at the gastric mucosal surface by Turnberg and coworkers (25, 190, 260) and by Silen and coworkers (230) and at the duodenal mucosal surface by Flemström and Kivilaakso (72, 136).

Several agents, including un-ionized weak acids and ethanol, damage the gastric mucosa and cause a marked increase in the disappearance of acid from the gastric lumen. Davenport (52) and others (20) provided evidence for damage-induced transmucosal leakage of interstitial and plasma HCO3 neutralizing intraluminal acid. This leakage, which should be distinguished from metabolism-dependent transport of HCO3 from the undamaged mucosa, was later found to be important in mucosal repair by maintaining a neutral pH under the mucoid cap formed on top of the repairing epithelium (119, 146, 213). The protective mucoid cap was shown to be primarily a thick fibrin gel layer formed from leaking plasma fibrinogen along with interstitial HCO3 and distinct from the adherent mucus gel layer covering the undamaged mucosa (210).

In this article we review the current state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared (see reviews, Refs. 12, 13, 66, and 78). Mucus is unique in the gastrointestinal tract in that the secretion, particularly in stomach, is a thick layer of gel adherent to the mucosal surface. The role of this gel layer in mucosal protection is a structural one: to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin reaching the underlying epithelium. Therefore, the emphasis on mucus in this review is on the form and function of this adherent mucus gel layer. The role of mucosal HCO3 secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal HCO3 secretion in this review is on the mechanisms and neurohormonal control of its secretion, the establishment of a surface pH gradient, and, in particular, the stimulation of HCO3 secretion by acid in the lumen, which is of primary importance, particularly in duodenum.


    ADHERENT MUCUS GEL LAYER IN SITU
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
A continuous layer of adherent mucus gel in stomach, duodenum, and more distal parts of the intestinal tract has been demonstrated using a number of methods in vitro (8, 36, 130, 131, 176) and in vivo (6, 24, 44, 111, 204, 238). In this context it should be emphasized that conventional histological methods using organic solvents and paraffin embedding result in shrinkage and dehydration of the mucus gel layer to give little or no extracellular mucus visible on mucosal sections (8). The original methods demonstrating an adherent gastric mucus gel circumvented these problems by either 1) indirectly measuring differences in refractive index with a slit lamp and pachymeter (36) or 2) directly observing unfixed mucosal sections on which the mucus gel appeared as a translucent layer of variable thickness (10–250 µm, in rats) between a clear bathing solution and the dense mucosa (131, 159). Subsequently, histological methods have been developed that preserve the surface adherent mucus layer with the use of cryostat sections, milder fixing conditions, and water-soluble mountants (18, 130). Mean mucus layer thickness values obtained with the use of these methods for antrum in humans and rats are 106 µm (minimum, 50 µm) and 166 µm (minimum, 90 µm), respectively (130, 168), and in rats approach values seen in vivo (24).

The paucity or absence of mucus on histological sections prepared using conventional procedures initially led to controversy as to whether a continuous layer of mucus existed over the gastric mucosal surface (166, 255). This was compounded by the misnaming, as mucus, of the mucoid cap seen on histological sections of reepithelializing gastric mucosa following acute damage (166). The mucoid cap on top of the damaged and repairing mucosa is primarily a fibrin gel with necrotic cells and remains of the adherent mucus layer from the original, undamaged mucosa (8, 210). This mucoid cap, in contrast to the adherent mucus gel, is three to four times thicker and can be preserved using standard histological fixation procedures.

An elegant method involving the use of intravital microscopy to observe adherent mucus gel layers on mucosal surfaces in vivo, initially developed for stomach, has now been applied throughout the gut of anesthetized rats (6, 24, 111, 197, 204, 238). With the use of this method, a continuous, translucent mucus gel layer can be seen on mucosal surfaces, with a mean thickness of 189 µm in corpus, 274 µm in antrum, and 170 µm in duodenum (24). What is particularly interesting is that two physical forms of mucus have been observed: a loosely adherent mucus layer that can be removed by suction and a firm adherent mucus layer that remains. This remaining firm adherent mucus is substantial and continuous in antrum, corpus, and colon, while it is very thin or absent in duodenum, jejunum, and ileum, where the primary function is absorption of nutrients (Fig. 1). In stomach in vivo, the firm adherent mucus layer presumably equates to the stable protective barrier, whereas the superficial loosely adherent layer, likely to be largely removed by the shear forces of the digestive process, would act more as a lubricant. The loosely adherent layer continually increases in thickness at a rate slowest in the stomach and greatest in the colon, where it is very thick (714 µm) and copious (24). Compatible with these observations in vivo is the recent isolation from pig stomach in vitro of two mucus secretions that differ distinctly in their gel rheology and, possibly, component mucin multimeric structure (242). There is a sloppy mucus gel that is readily broken down by low applied shear forces and an underlying firm adherent mucus gel that is resistant to applied shear and collapses only at shear forces two orders of magnitude higher.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Thickness of mucus gel layers along the rat gastrointestinal tract. The gastrointestinal tissues of thiobarbiturate-anesthetized rats were mounted luminal side up for intravital microscopy, and mucus thickness was measured before and after partial removal by suction. A loosely (sloppy) adherent mucus layer was removed by suction to leave a firm adherent mucus layer attached to the mucosa. This loosely adherent mucus layer was continuous and did not follow the contours of the villi in the intestine. In stomach and colon, the firm adherent mucus layer was continuous, but in the small intestine, this firm mucus layer had a patchy distribution and was absent on individual villi. [Adapted from Atuma et al. (24).]

 
An alternative approach, the application of confocal microscopy and fluorescent beads to observe the surface of the adherent mucus layer, has been used in anesthetized rat and mouse stomach in vivo (29, 44). Mucus layer thickness values (median, 50–75 µm) obtained for rat corpus by using this approach are ~30–40% of those obtained using intravital microscopy. This difference, in part, could be due to the use of a perfusion system in the confocal studies, likely to remove the loosely adherent mucus gel, whereas unstirred conditions pertain to intravital microscopy studies (24, 44). At the same time, mucus layer thickness values from confocal microscopy are still noticeably lower than those for the firm mucus layer (i.e., after suction) from intravital microscopy studies. A comparative study in vivo using both methodologies for observing mucus thickness and at varying rates of perfusion would be interesting.


    STRUCTURE AND STABILITY OF MUCUS LAYER
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Two factors that determine the efficacy of the adherent mucus layer as a barrier are, first, the gel structure, on which depend the stability and permeability of the adherent mucus layer, and second, the thickness of the adherent layer (see below). Compared with other gastrointestinal secretions, the adherent mucus gel form is physically unique, and an understanding of its structure and properties is essential to underpin physiological studies. Rheological studies have shown that adherent gastrointestinal mucus gels from stomach, duodenum, and colon are all well-defined viscoelastic gels that do not dissolve on dilution (8, 17, 30, 31, 211). Mucus secretions as gels are also unique in that they flow over a relatively long time (30–120 min), reannealing when sectioned. It is this property that distinguishes mucus gels from other noncovalent but rigid gels (e.g., agar). In functional terms, these flow properties are key to the adherent mucus gel layer forming a continuous cover over the mucosa in vivo. Mucus gels are stable, and exposure of isolated gastric mucus gel to pH 1–8, hypertonic salt (e.g., 2 M NaCl), or bile does not disperse the gel or affect its rheological properties (30, 31, 211). The mucus gel is dissolved by reduction with thiol agents or proteolysis, both of which destroy the multimeric structure of the component mucins (19, 31).

Mucus secretions from all regions of the gut have the same generic gel structure (8, 31, 211), and this is reflected by common structural patterns in the component gel-forming mucins. The central regions of all gel-forming mucin units have a protein core covered by a sheath of glycan chains that are flanked at both ends by nonglycosylated protein domains rich in cysteine. The cysteine-rich domains are the location of the interchain disulfide bridges between the mucin units that are polymerized end to end into large mucin multimers (7, 8, 19, 91, 155, 183). Gel-forming mucins are very large molecules (reported molecular masses of 5–45 x 106 Da) consisting of multimers of mucin units (2–3 x 106 Da). The peptide core alone of each mucin unit contains >5,000 amino acids, around which are packed glycan chains that comprise the major portion by weight of the molecule (between 50 and 80%). Until recently, progress in this field was slow, primarily because of the structural complexity of the component mucins and the difficulties of performing meaningful rheological studies of mixtures of such molecules. The structure of each of the four gel-forming mucins, MUC2, MUC5AC, MUC5B, and MUC6 (published only in part), has now been elucidated with cloning and sequencing of the mucin genes. This has led, over the past decade, to an explosion of information on the complexities of mucin structure, biosynthesis, and secretion, and there are many reviews available (8, 38, 46, 47, 83, 88, 132, 164, 183, 252). Three of the four gel-forming mucins are well expressed in the normal gastrointestinal tract. In the stomach, MUC5AC is expressed by the surface mucous cells of cardia, fundus, and antrum, and MUC6 is expressed by the mucous neck cells of fundus and of antral glands in cardia and antrum (54, 103, 105). MUC2 is expressed by goblet cells from duodenum to colon, whereas MUC6 is expressed by Brunner's glands in duodenum (103, 105).

The general features of mucus gel structure are known, although there are many questions remaining. The multimeric structure (previously referred to as polymeric structure) of the mucins is essential for gel formation (7, 19, 31, 220). Gel strength and stability have been shown to relate directly to the percentage of multimeric mucin, relative to the monomeric form, in the gel (211). In gastric ulcer and Helicobacter pylori infection, the percentage of multimeric mucin in the adherent gastric mucus layer falls, indicative of a weaker and less stable mucus gel (10, 168, 264). The large, highly hydrated multimeric mucin molecules interact noncovalently to form the mucus gel network (8, 17), but the chemical nature of these interactions is unclear. Recent rheological studies point to a complexity of different, noncovalent interactions of a variety of types and strengths between the mucin molecules (241). More than 90% of these interactions are transient (i.e., make and break over a finite time), explaining the unique flow properties of mucus gels.

What has emerged from such studies is evidence that gel-forming mucins from different secretions or even the same secretion, while possessing a common structural pattern, show many specific structural differences in their protein core, glycan side chains, antigenic properties, and negative charge (8, 38, 46, 54, 83, 132). Furthermore, histological studies suggest that there maybe different structural patterns within the adherent mucus layer itself. Laminated layers of surface mucus and glandular mucus, which stain for different glycan structures, have been observed in the adherent gastric mucus layer in situ in humans (100, 176). Alternating layers of MUC5AC and MUC6, the two gastric mucin gene products, have been reported in the mucus layer in sections of human gastric mucosa (104). Moreover, recent studies using the novel technique of high-pressure freezing/freeze substitution, which preserves intact not only the mucus gel but also the fluid luminal phase, showed a triple lamination of different mucin structures within the secreted mucus gel layer at the mouth of the gastric pits (203). In the present context, the key question for the future is whether and to what extent these subtle structural differences, between and within the different mucin secretions, influence gel structure sufficiently to effect the stability and permeability of the adherent mucus gel layer.


    TREFOIL PEPTIDES AND THE ADHERENT MUCUS LAYER
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
There is considerable interest in a putative structural role in mucus gel for the cosecreted, low-molecular-weight trefoil peptides. Three trefoil peptides have been identified in humans and have been shown to be key factors in stimulating cell migration and promoting epithelial repair after damage (59, 106, 185, 202, 240). TFF1 is cosecreted with MUC5AC mucin in stomach, TFF2 with MUC6 mucin in glands of stomach and duodenum, and TFF3 with MUC2 mucin from goblet cells. In TFF1-null mice, the antral and pyloric gastric mucosa exhibit severe hyperplasia and are almost entirely devoid of mucus, showing that expression of the latter is dependent on the former (150). TFF1 is present in high levels in the adherent gastric mucus gel layer and is strongly, noncovalently bound to isolated mucin (167). In a yeast two-hybrid system, TFF1 interaction with the disulfide-bridging von Willebrand C domains of MUC5AC and MUC2 has been shown and interpreted as facilitating multimerization of the mucin. However, these interactions have yet to be confirmed by in vitro studies (248). In HT-29 cells, upregulation of trefoil factor secretion is stimulated by mucin secretagogues, and the former is bound to mucin in the adherent mucus layer (92). There are various studies suggesting that trefoil peptides influence mucus gel properties. Thus rat gastric or intestinal mucin acts cooperatively with TFF3 in the attenuation of damage to a human T84 colonic cancer cell line by a variety of agents (133). TFF2 addition dose-dependently decreased the rate of diffusion of H+ through a 5% solution of pig gastric mucus and slowed the initial acidification rate of gastric mucosal cells in vivo (237). These results were interpreted as interaction of TFF2 with the mucus gel, decreasing permeability of the latter to protons. Human TFF2 has been shown to increase the viscosity of a commercial preparation of pig gastric mucin, leading to formation of a gel, and this was interpreted as evidence of trefoil peptides interacting with the mucins to stabilize the gel network (246). However, it is difficult to relate these in vitro studies to the adherent gel secreted in vivo, because commercial pig gastric mucin preparations are substantially degraded and non-gel forming (8). Clearly, trefoil peptides are an integral part of the intracellular mucus secretory vesicles and the ensuing adherent mucus gel; furthermore, they interact strongly with the component mucins. However, it is still an open question as to whether trefoil factors have a structural role in the secreted mucus gel in vivo or the latter primarily provides a stable extracellular support for the former, similar to that for secretory IgA. Trefoil peptides may well play a role in the intracellular assembly and/or packaging of mucins.


    FACTORS INFLUENCING MUCUS LAYER THICKNESS
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
A primary factor in determining the protective efficacy of the mucus barrier in vivo is the thickness of the adherent firm mucus layer. Thickness of the mucus layer is the result of a dynamic balance between its secretion and its erosion mechanically by shear forces of the digestive process and by proteolytic degradation, particularly from luminal pepsin in stomach (8, 9). There is a wealth of work on secretion of the component mucins, the intracellular control of this process, and the response to neural, hormonal, and paracrine stimulation, as well as the effect of inflammatory mediators. A variety of in vitro and in vivo models, including cell culture, have been used in these studies, and there are many reviews (8, 46, 82, 83, 239, 252). It is difficult, however, to relate these studies on mucin secretion directly to the effective thickness of the adherent mucus gel in vivo, because of variables such as mucin concentration in the gel, rates of mucus erosion in vivo, and proportions of sloppy (loosely adherent) and firm mucus in the secreted product. Where gastroduodenal mucus thickness has been measured directly, it has been shown to be increased by hormonal, paracrine, and neural stimulation and, in duodenum, by topical acid.

In rat stomach in vivo, mucus layer thickness, measured on unfixed mucosal sections, is increased up to threefold by topical prostaglandins, carbachol (intraperitoneal), and secretin (intravenous) (8, 9, 131, 159). Rat gastric mucus layer thickness was unchanged after exposure to acid pH 1 or 2 over 3 h in vivo (16). Direct observation in rat stomach in vivo showed no change in adherent mucus layer thickness over time in the presence of acid pH 1.7, whereas pentagastrin (intravenous), cimetidine (intraperitoneal), and prostaglandin E2 (PGE2) all increased mucus layer thickness by up to one-third (170, 171). Further evidence that exposure to luminal acid does not change gastric mucus layer thickness was provided by reports that no significant increase in thickness occurred in pentagastrin-stimulated rats in vivo, with or without inhibition of acid secretion by omeprazole (204). Central vagal stimulation by injection of thyrotropin-releasing hormone analog RX 77368 increased gastric mucus layer thickness by up to 30%, and this was unaffected by acid pH 1, indomethicin, or omeprazole (238).

In rat duodenum, in contrast to rat stomach, exposure to luminal acid does increase mucus layer thickness, and this is mediated via extrinsic sensory nerve fibers that release CGRP followed by stimulation via nitric oxide (NO) and cyclooxygenase (COX). The rate of continuous replenishment of duodenal mucus (sloppy) in rat in vivo, following its removal by suction, was increased (44%) by exposure to HCl pH 2 for 10 min and inhibited by indomethacin or NO synthase inhibitor N{omega}-nitro-L-arginine (197). More recently, rat duodenal mucus layer thickness in vivo was shown to be increased by >50% in response to luminal acid, primarily by stimulation of a capsaicin-sensitive pathway including NO, vanilloid receptors, and afferent nerves (3, 6). PGE2 (luminal or intravenous) similarly increased duodenal mucus layer thickness, but, interestingly, indomethacin also abolished the acid/capsaicin-mediated response, suggesting that COX pathways may be the common mechanism for duodenal mucus secretion. Once the acid stimulus is withdrawn, the mucus layer thickness values returned to basal levels of the order of 100 µm (3), indicating that the increase may have reflected primarily changes in sloppy mucus gel, which is subsequently removed by the shear forces of the perfusion system used. The acid-induced stimulation of duodenal mucus layer thickness in the duodenum is part of a series of mucosal protective mechanisms in response to increased luminal acidity, including increased HCO3 secretion and blood distribution (69, 94, 114).


    MUCUS AND PROTECTION AGAINST PEPSIN
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Pepsin, in contrast to acid, has received relatively little attention as the other endogenous aggressor in gastric juice. Yet, in an anesthetized rat stomach model, mildly excess pepsin (2 mg/ml, maximal secretory levels in humans over 2 h) causes extensive mucosal damage under conditions in which acid pH 1 or 2 alone is ineffective (11, 16). Pepsin damage is characterized by focal areas of discontinuity in the adherent mucus gel layer, localized hemorrhagic punctuate ulcers with bleeding into the lumen, and no evidence of reepithelialization or mucoid cap formation. Damage by pepsin is markedly different from that caused by ethanol (70%, 45 s) or 2 M NaCl (14, 16, 210, 213). These agents rapidly penetrate the mucus barrier, resulting in exfoliation of the epithelial layer with a dramatic increase in mucosal permeability, followed by reepithelialization under a fibrin-based mucoid cap. Pepsin digestion of the adherent mucus layer on Necturus gastric mucosa, bathed in acid pH 2.5, resulted in a fall in pH at the epithelial surface from pH 5.22 to pH 2.35 over 2 h (137). In esophagus, where squamous epithelium is devoid of a significant adherent mucus gel layer (56), evidence from animal models (247) and clinically in humans (102) points to pepsin rather than acid as the critical factor in gastroesophageal reflux damage.

The adherent mucus gel layer is a physical barrier to luminal pepsin accessing the underlying mucosa. The rate of diffusion of solutes through mucus gel decreases progressively with increasing molecular size (14, 16, 27, 29, 55), and it takes several hours for pepsin (pH 7.0) to permeate a thin layer of mucus gel in vitro (11, 14). The physical explanation for this is that measured rates of diffusion of macromolecules in unstirred solution are very slow, of the order of 1 x 10–6 cm2/s, and calculations show that it would take hours for pepsin (32 kDa) to diffuse through the adherent gastric mucus layer in vivo (11). There also may be some steric hindrance to macromolecular permeability through a 5% mucus gel. In duodenum in vivo, controlled removal of the thin mucus layer by papain digestion markedly enhanced the HCO3 secretory response to cholera toxin (85 kDa), H. pylori Vac A toxin (89 kDa), and glucagon (3.5 kDa) (68). These studies show that even the thin layer of firm mucus gel in the duodenum in vivo limits access of larger-molecular-weight toxins to the mucosa. They also provide a note of caution in secretory studies involving luminal application of such agents. Interestingly, in these studies, removal of the mucus layer also enhanced the stimulation of HCO3 by PGE2 (335 Da), but this effect is most likely explained by the binding of the relatively small amounts of secretagogue to mucin (68).

Because luminal pepsin cannot permeate the continuous adherent mucus layer within a physiologically meaningful time scale, it follows that the latter is an effective barrier, probably the major barrier in vivo, against proteolytic digestion of the underlying epithelium (14, 16). At the same time, luminal pepsin at acidic pH slowly hydrolyzes and erodes the adherent mucus layer. However, at the levels of luminal pepsin in vivo, this is normally balanced by new secretion (9, 14, 31). Lack of interest in pepsin as a mucosal damaging agent has been due primarily to the pharmaceutical success of acid inhibition in peptic ulcer treatment and the absence of a good selective inhibitor of pepsin secretion. The proteolytic activity of pepsin in gastric juice on an average protein substrate falls rapidly above pH 3 (102, 181), and it has been assumed that above this pH most, if not all, of the pepsin activity in vivo is lost. However, depending on the substrate and pepsin type, there can be significant pepsin activity well above pH 3. Thus pepsin 1, which rises fivefold in peptic ulcer disease to reach 20% of the total activity in gastric juice (243), has substantial mucolytic activity between pH 3 and 5 (14, 181). Levels of serum pepsinogen 1 and 2 have been shown to be increased in H. pylori infection and peptic ulcer disease, whereas low serum pepsinogen levels are a good indicator of atrophic gastritis (35, 201). However, the relationship between serum pepsinogen levels and luminal gastric pepsin activity has yet to be clarified experimentally. Pepsin-induced mucosal damage and activity of the secreted individual pepsin isoforms in peptic ulcer disease and gastroesophageal reflux disease, as well as how luminal pepsin activity relates to serum pepsinogen levels, all merit further study.


    PH GRADIENT AT THE GASTRIC MUCOSAL SURFACE
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
The conventional view is that the primary role of the adherent mucus layer in protection against acid is to form a stable, unstirred layer at the mucosal surface that prevents immediate mixing of the secreted HCO3 with the excess of acid in the lumen. The mucus gel, by acting as a mixing barrier, enables the stabilization of a pH gradient from acid in the lumen to near neutral at the mucosal surface (11, 13, 78). Direct evidence of the presence of a surface pH gradient on top of gastric mucosa comes from numerous demonstrations of a pH gradient across the mucus layer. This has been shown for human (25), rabbit (260), and amphibian gastric mucosa in vitro (137, 230) and for rat (184, 204, 227), canine (179), and human stomach in vivo (190, 212). Various other studies support such a role for the mucus layer; for example, in Necturus stomach, in vitro removal of the mucus layer by pepsin or N-acetyl-cysteine causes intracellular and juxtamucosal surface pH to fall on perfusion of luminal acid pH 2.5 (137). There is an inverse correlation between adherent mucus layer thickness and the initial acidification rate of mucosal cells in rats in vivo (57). In mouse stomach in vivo, mucus has been shown to strengthen the surface unstirred layer effect (29). Also, a collapse of the alkaline surface pH gradient, which would otherwise inhibit pepsin activity, must occur when luminal excess of this enzyme causes loss of the adherent mucus layer and digestion of the underlying mucosa in vivo (16).

Under conditions of acid secretion and HCl (pH 1) instilled into the lumen, a pH gradient across the gastric mucus layer is maintained with a pH of >7 at the mucosal surface (Fig. 2) (184). Other workers using a similar system in rat and mouse stomach, but with confocal imaging and the fluorescent dye Cl-NERF, demonstrated a surface pH gradient with superfusion at luminal pH 3.0 (28, 44, 49). Furthermore, in this system, surface and intraglandular alkalinity were increased by luminal administration of dimethyl PGE2, a compound previously shown to stimulate both gastric and duodenal mucosal HCO3 secretion (23, 65, 235). Also, at luminal pH 3.0, the relatively alkaline surface pH 4.3 ± 0.1 was acidified by indomethacin (to pH 3.6 ± 0.2), and subsequently dimethyl-PGE2 restored surface pH to 4.2 ± 0.2 (28). The authors concluded that the preepithelial alkaline layer in the mouse stomach is regulated by endogenous COX activity. Similar increases in surface alkalinity in response to E-type prostaglandins have been observed in rat stomach (195).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Gastric juxtamucosal pH in pentagastrin- and ranitidine-treated rats in vivo. The mucus layer was left intact, and juxtamucosal (mucoepithelial interface) pH was measured with H+-sensitive microelectrodes. After a 15-min control period with isotonic saline, the gastric lumen was exposed to 100 mM HCl for 10 min, as indicated. This was followed by a second control period with isotonic saline. Note the only slight and transient decline in juxtamucosal pH in animals treated with pentagastrin (40 µg·kg–1·h–1 iv, starting 30 min before acid exposure) to stimulate parietal cell acid secretion. In contrast, there was a marked decline in juxtamucosal pH in animals treated with the histamine H2 antagonist ranitidine (1 mg/kg iv, 30 min before acid exposure) to inhibit parietal cell acid secretion. [Adapted from Phillipson et al. (184).]

 
Results with the noninvasive confocal imaging technique at luminal pH 3.0 are thus broadly in line with those recorded on penetration of the mucus layer with pH-sensitive microelectrodes at higher luminal acidities of pH 1–2. However, with superfusion at luminal pH 5.0 in both rat and mouse stomach (28, 44, 49), the surface pH gradient was shown to be reversed with a more acidic juxtamucosal pH. This acidity was enhanced by pentagastrin and eliminated by omeprazole, indicating its dependence on acid secreted by the parietal cells. Moreover, it was associated with marked variations of surface pH with the rate of luminal perfusion. A higher juxtamucosal pH at luminal pH 3.0 compared with that at luminal pH 5.0 could reflect higher gastric HCO3 export stimulated by the higher luminal acidity (28). A possible explanation for the reversal of the pH gradient observed at pH 5 is that the zone of CO2 released within the gastric mucus gel, during neutralization of H+ by HCO3, moves closer to the epithelial surface at lower (less stimulated) rates of HCO3 secretion (62, 87, 244). The dependence of gastric HCO3 export on interstitial/blood HCO3 supply (see below) also may influence the juxtamucosal pH. Further and comparative studies with pH-sensitive microelectrodes and confocal imaging, particularly above luminal pH 3 and under different rates of perfusion, are needed to clarify the dynamics of the pH gradient at the gastric surface.

The adherent mucus layer per se is not a diffusion barrier to acid that diffuses rapidly through the gel matrix both in vitro and in vivo. Numerous studies have measured diffusion rates of H+ through fresh preparations of gastric mucus gel in vitro and have shown that after initial saturation of binding sites, such diffusion rates are retarded 4- to 10-fold compared with those in an equivalent "unstirred" layer of solution (57, 169, 236, 259). These results have been interpreted to show that mucus slows the mobility of H+ by acting as a barrier to its free diffusion. Various possible mechanisms for this effect have been reviewed (57). However, calculations from these measured rates of diffusion of H+ in vitro indicate that acid would still rapidly permeate a layer of adherent mucus gel 100–200 µm thick in a matter of minutes in vivo (11). Studies in vivo confirm that the adherent mucus gel layer is not a significant permeability barrier to H+ (as opposed to a stable unstirred layer and mixing barrier). Thus acidification of juxtamucosal surface pH, as monitored by electrodes, can occur very rapidly through the mucus layer when acid is applied to the luminal side (137, 179, 184) (Fig. 2). Recently, elegant experiments in mouse stomach in vivo showed that the rate of diffusion of the low-molecular-mass (400 Da) fluorescent dye CL-NERF through the mucus is the same as that through an unstirred layer of luminal solution (29). This provides clear evidence that, in vivo, the adherent mucus layer is freely permeable to ions and small-sized molecules.

There have been suggestions that mucus undergoes a conformational change at pH ~4 that affects its permeability properties with the result that it becomes more impermeable to H+ at higher levels of luminal acidity. The evidence to support this comes from numerous different studies on mucin solutions involving viscosity (34), viscous fingering (33), and light scattering (41). The evidence from in vivo studies in rat and mouse stomach and Necturus stomach in vitro does not support such pH effects on H+ permeability through mucus in vivo (29, 137, 179, 184). Furthermore, rheological results obtained with mucin solutions cannot necessarily be extrapolated to the native gel, where mucin molecules are restricted by interactions in a gel network and may not undergo the conformational changes seen in free solution, particularly where changes in ionic strength are involved. Thus the rheological characteristics of intact mucus gel are unaffected by exposure to low ionic strength or acid down to pH 1 (31, 211), yet both of these conditions result in a dramatic increase in mucin solution viscosity (17, 34, 41).

Considerable interest has been generated in the role of lipids within the adherent mucus layer providing a barrier to H+ (101, 153, 154). Evidence for a lipid barrier comes primarily from high contact angle measurements over the gastric mucosa, indicating a hydrophobic surface and the presence of lipids, including surfactants, in adherent gastric mucus. Loss of mucosal hydrophobicity, measured as sharp decreases in contact angle, and decreased phospholipid occur after exposure to known mucosal barrier breakers (e.g., bile and aspirin) (90, 154, 156). The reverse is seen with EGF, which is reported to increase mucosal resistance to acid (157). How lipids in mucus could form a barrier to acid is unclear, although monolayers or multilayers of phospholipid molecules within the mucus layer have been proposed (101, 154). Furthermore, it is uncertain to what contact angle measurements refer. Full drying times of 40–50 min are necessary to obtain consistent results (223, 224), and during this process the adherent mucus gel is progressively dehydrated (11). Even if a lipid barrier does exist in the adherent mucus layer, the evidence of the free permeability to H+ through mucus in vivo (discussed above) demonstrates that it is not effective against acid.


    ROLE OF THE ALKALINE TIDE IN GASTRIC MUCOSAL PROTECTION
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Luminal acidities below pH 2.0–3.0 have been reported to dissipate the gastric mucosal surface pH gradient with consequent exposure of the epithelial cell surface to the acidity of the bulk luminal solution. However, this reduction of surface pH occurred in stomachs in which gastric mucosal secretion of HCl was low or absent. As illustrated in Fig. 2, recent work (184, 227) has shown that the pH gradient in the surface mucus gel is markedly resistant to luminal acid pH 1.0 after stimulation of gastric acid secretion by pentagastrin or the histamine H2-receptor agonist impromidine. In contrast, there is an acidification of the surface mucus gel during inhibition of mucosal HCl secretion by the histamine H2-receptor antagonist ranitidine. The maintenance of the surface pH gradient in acid-secreting stomachs can be explained by the increased supply of HCO3 released to the interstitium from the HCl-secreting parietal cells, i.e., the "alkaline tide," carried to the surface mucosa by the gastric mucosal vasculature (65, 86).

The importance of plasma/interstitial HCO3 in protecting the gastric mucosa against acid-induced damage was first demonstrated in dogs (51) and rats (135) in vivo, where parenteral administration of exogenous HCO3 prevented ulceration of the acid-exposed gastric surface. Furthermore, serosal (basolateral)-side administration of HCO3 has a similar acid-protective action in amphibian (137, 138) and rat (191) mucosa in vitro. Studied in the amphibian preparation, serosal-side administration of other buffers, e.g., HEPES, did not protect the mucosa, and pretreatment with DIDS, or removal of serosal-side Na+, prevented the protective action of serosal-side HCO3. The combined results provide strong evidence that uptake of HCO3 by basolateral Na+-HCO3 cotransport enhances alkaline secretion by the surface epithelial cells (65), resulting in an increased ability of the gastric epithelial surface to resist luminal acid. The presence of HCO3 in the serosal-side solution furthermore enhances the rapid reconstitution of epithelial integrity in damaged gastric mucosa in vitro (213).


    SECRETION OF ACID AND PEPSIN FROM GASTRIC GLANDS ACROSS THE MUCUS LAYER
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Acid and pepsin, secreted very rapidly from the gastric glands upon stimulation, must gain access to the stomach lumen across a continuous adherent mucus layer. The best explanation is that gastric juice transverses the mucus layer though channels under pressure from the glands (8, 111). Compatible with such an explanation for acid is that pH measurements in vivo under secretory conditions do not show lateral diffusion of H+ away from the gastric glands at the mucosa-mucus interface (111, 184, 204). Also, it is difficult to explain how pepsin could transverse the mucus layer if it were not through channels. Channels containing acid in mucus layers impregnated with the acid-sensitive dye Congo red have been observed via direct microscopy in secreting rat stomach in vivo (111, 127). These Congo red-stained, 5- to 7-µm-wide channels of acid arise from crypt openings, appear to be discrete structures in that they remain after transient inhibition of acid secretion, and are formed during the secretion process (127). Congo red precipitates at acid pH, and it is not yet clear whether these channels naturally have a defined structure within the mucus gel or, alternatively, are formed in some way by interaction of the mucus gel with Congo red. In the same system, high intraglandular pressures (5–20 mmHg) in the gastric glands during acid secretion have been demonstrated, and this would provide a driving force for pushing secretions through the mucus layer into the lumen (110, 225, 226, 228). An in vitro analogy has been made with the phenomenon of viscous fingering, where a discrete boundary is observed between a high-viscosity mucus solution and low-viscosity HCl injected under pressure into it (27, 33).

Other studies have failed to show acid channels in the adherent mucus gel during acid secretion. With the use of confocal microscopy and the fluorescent acid-sensitive dye CL-NERF, no channels were observed in the mucus layer in secreting rat stomach in vivo (44), despite the spatial resolution of the technique and its demonstrated ability to measure differences in pH between glandular pits and the surrounding epithelium. Why channels are not seen with confocal imaging but are seen with direct microscopy is not yet clear, and comparative experiments using both methodologies in the same preparation would help resolve this. In another study in vitro, with the use of sensitive, double-barreled microelectrodes, no evidence was found for acid channels in the mucus layer of acid-secreting guinea pig mucosa (206). From this study, a model has been proposed in which protons transverse the mucus gel layer buffered by the mucin and are released from the latter by conversion of pepsinogen to pepsin at the lower pH values nearer the lumen (206, 207). There are difficulties with this model in that it would require rapid turnover of the firm mucus gel layer at a substantially much greater rate than that seen in vivo (24). Also, pepsinogen is converted to pepsin autocatalytically at acid pH, and it is likely that it will be, almost entirely, the activated enzyme that emerges from the gastric gland. Furthermore, the observed stability of the gastric mucus gel to acid in vitro and in vivo is not compatible with the postulate that it is widely impregnated with pepsin or pepsinogen, which under conditions of low pH would result in mucolysis (16, 31).

Finally, secreted mucosal HCO3 presumably diffuses freely from the surface of the gastric epithelium through the mucus gel layer. Because the alkali secretion (HCO3 export) by the gastric mucosa in the main reflects a Cl/HCO3 exchange process in the apical membrane of the surface epithelial cells (see below), an association between gastric crypt secretion, volume flow, and alkali secretion would appear unlikely.


    GASTRIC BICARBONATE SECRETION
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
In frog gastric mucosa mounted as sheets in Ussing chambers (61, 75), characteristics of the HCO3 secretion by antral mucosa, devoid of parietal and chief cells, and fundic mucosa point to the surface epithelium, common to both preparations, as the origin of the secretion. Similar characteristics have been seen for fundic and antral alkaline secretion in conscious dogs with pouches (142, 143). Studies of frog (66, 231) and Necturus mucosa (137, 138) show Na+-HCO3 cotransport at the basolateral membrane as the major mechanism for import of HCO3. Recently, it was demonstrated that expression of the Na+-HCO3 cotransporters NBC1 and NBC2 in rabbit stomach is at a higher level (5.5- and 2.5-fold higher, respectively) in the mucus containing surface cells than in the parietal cells (196). The inhibition of alkaline secretion in vitro and in vivo by carbonic anhydrase inhibitors, although at relatively high doses (61, 75, 193), does suggest that some HCO3 is also formed intracellularly from CO2 and H2O. Secretion of HCO3 by frog fundic mucosa in vitro is dependent on luminal but not serosal Cl (63) and is, in contrast to that of H+ transport, not associated with changes in the transmucosal electrical potential difference (61). Recent immunological staining of rat and rabbit gastric mucosa demonstrated expression of a Cl/HCO3 exchanger of the SLC4 family, termed anion exchanger isoform 4 (AE4), in the apical membranes of gastric surface epithelial cells (262). These results provide strong evidence that Cl/HCO3 exchange is indeed a HCO3 export process in the gastric mucosa. Interestingly, recent work (48) also suggested an association between nonacidic Cl secretion and HCO3 secretion.

Recordings of pH within the lumen of the gastric glands have provided further information on the secretion of HCO3 by the gastric mucosa. Intraglandular pH in acid-inhibited, cimetidine-pretreated sheets of frog (Rana esculenta) fundic mucosa was measured with double-barreled pH glass microelectrodes inserted into the glandular lumen (53). The fluid was found to be slightly more alkaline than the bathing solution, and, interestingly, both carbachol and gastrin increased transport of alkali into the intraglandular lumen. The authors proposed that oxyntopeptic cells contribute to the gastric alkaline secretion in this species. In another study using double-barreled pH-sensitive microelectrodes with guinea pig fundic mucosa (206, 207), there was a gradient of increasing pH along the crypt lumen from 3.0 in the parietal cell region to 4.6 in crypt outlets. However, it should be stressed that the rate of acid secretion by mammalian gastric mucosa in vitro usually is much lower (≤5%) than that in vivo. This difference in rates may reflect insufficient oxygenation of the circulation deprived mammalian tissues in vitro. Furthermore, oxygen-deficient mucosal cells may swell and release mucus. The pH-sensitive indicator Lysosensor yellow-blue was used for measurement of net acid transport within isolated gastric glands in a recent elegant study (182). From the transient nature of the response to carbachol, it was proposed that this secretagogue may induce intraglandular secretion of alkali and/or proteins, such as pepsinogens and mucins, from chief and other nonparietal cells, that may buffer acid in the intraglandular lumen. An only slightly acidic intraglandular pH (pH ~5.3) was found upon confocal microscopy of rat fundic mucosa in vivo, and treatment with dimethyl-PGE2 increased intraglandular pH (49). E-type prostaglandins, which stimulate both gastric and duodenal alkaline secretion (65, 235), are also well-known inhibitors of gastric H+ secretion (256). These combined studies of intraglandular pH provide evidence that some HCO3 and/or acid-buffering substance originates from cells within the gastric glands, but this is unlikely to be quantitatively significant in net gastric mucosal HCO3 secretion in vivo.


    DUODENAL ALKALINE SECRETION
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
In all amphibian and mammalian species tested, the duodenal mucosal surface enterocytes secrete HCO3, and they do so at higher rates (per unit surface area) than does the stomach or more distal small intestine (43, 61, 64, 99, 143, 173, 214). Secretion of HCO3 by the duodenum was originally observed in proximal duodenum in dogs, and the submucosal glands (i.e., Brunner's glands) were then assumed to be the origin (65, 78). However, secretion by Brunner's glands could not be separated from that of the mucosa itself in early experiments. Brunner's glands are unique to mammalian species and are confined mainly to the submucosa. Secretory units consist primarily of a mucin-producing cell type, and in addition to mucus, these cells are known to secrete epidermal growth factor and trefoil peptides (144). Video microscopy of the acinar lumen of Brunner's glands in guinea pig duodenum in vitro demonstrated secretory responses to cholinergic vagal fibers but not to capsaicin-sensitive or enteric intrinsic nerves (165). Secretion of a limited amount of HCO3 by Brunner's glands cannot be excluded in some species, but this has not been experimentally confirmed, and micropuncture studies of the gland or duct lumen need to be done.

Knowledge about the HCO3 transport processes in the duodenal epithelium per se originates from numerous studies (65, 69) involving animals and humans in vivo, amphibian and mammalian mucosa in vitro, isolated mammalian duodenal enterocytes, isolated cell membranes, and, more recently, genetically modified mice. Microfluorospectrophotometric studies of mixed villous and crypt enterocytes isolated from proximal duodenum and loaded with the pH-sensitive fluoroprobe BCECF (117) confirmed previous hypotheses based on studies of amphibian mucosa in vitro (66), namely, that duodenal enterocytes possess at least three mechanisms for acid-base transport (Fig. 3). These are 1) amiloride-sensitive Na+/H+ exchange, which extrudes acid; 2) duodenal enterocytes, which import HCO3 at the basolateral membrane by Na+(n)-HCO3 cotransport and export HCO3 via an apical anion conductive pathway and Cl/HCO3 exchange; and in addition, 3) paracellular migration of HCO3, which is dependent on transmucosal hydrostatic pressure in vitro (64) and intestinal motility in vivo (172, 174). Studies of duodenum from knockout mice deficient in the cystic fibrosis transmembrane conductance regulator (CFTR) (45, 93, 107, 129, 209) provide strong evidence that CFTR is the membrane-spanning conductance transporting HCO3 as well as Cl (Fig. 3). CFTR is presently considered a principal anion channel pathway in Cl and HCO3 export in airway and intestinal epithelia, but it also exerts several cellular actions independent of channel activity (32). Endoscopic specimens mounted in Ussing chambers have been used in a study of CFTR activity in human duodenum (186). In line with findings from mouse duodenum, basal HCO3 secretion and short-circuit current were significantly lower in biopsies from patients with cystic fibrosis than in those from normal subjects. Present evidence thus strongly suggests that CFTR is the important apical conductive pathway in agonist-stimulated duodenal mucosal HCO3 secretion mediated by cAMP as well as by cGMP and intracellular Ca2+ concentration ([Ca2+]i) (22, 45, 107, 129, 209, 222). Basal HCO3 secretion, in contrast, seems mainly dependent on apical Cl/HCO3 exchange (222, 254). Recently, PAT1 (SLC26A6) was identified as a major Cl/HCO3 exchanger in the apical membrane of the duodenum (257), along with the DRA (SLC26A3) Cl/HCO3 exchanger (121, 161).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Duodenal acid-base transporters. The model is based on studies of isolated duodenal enterocytes, membrane vesicles, and duodenal mucosa in vitro and in vivo in several species and, more recently, from studies of genetically modified mice. Species differences, if any, seem small, but the mucosal location of the transport processes (villus and/or crypt) has not yet been fully determined. HCO3 reaches the epithelium via blood as well as secondary to the intracellular carbonic anhydrase (CA) conversion of CO2 + H2O. HCO3 is imported via Na-HCO3 transport and exported via an apical conductance (CFTR) pathway and Cl/HCO3 exchange. In addition, there is paracellular migration of HCO3, dependent on intestinal transmucosal hydrostatic pressure and motility. Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 are all reported to be expressed and contribute to intracellular pH regulation in duodenal enterocytes. Evidence for electrogenic as well as electroneutral Na-HCO3 cotransporters (NBC) has been presented. Any factors that diminish HCO3 export may increase the vulnerability of the mucosa to acid injury. AE, ion exchanger.

 
Parallel operation of CFTR Cl channels and apical Cl/HCO3 exchangers is a possible mechanism for cellular export of HCO3 (251). The CFTR channel would provide Cl to the luminal surface and act as a Cl leak pathway, preventing intracellular accumulation of Cl. However, it was found in rabbit and rat duodenum in vitro that Cl/HCO3 exchange is neither necessary nor increased upon cAMP-induced stimulation of mucosal HCO3 secretion (222). In contrast, CFTR-dependent Cl/HCO3 exchange does occur in cAMP-stimulated mouse duodenum (45). The difference between species may reflect relatively greater rates of secretion in the mouse duodenum, a thinner preparation that would seem easier to maintain well oxygenated in vitro. It should be noted that uroguanylin, inducing cGMP-mediated HCO3 secretion, also causes some increase in transmucosal short-circuit current in duodenum from CFTR knockout mice (129). This may suggest cGMP-induced activation of a minor electrogenic pathway, obscured in wild-type animals by the larger CFTR conductance.

It has been believed that intestinal secretions are of crypt origin, whereas absorptive functions reside in villi. Interestingly, recent work (21, 22) suggested involvement of the CFTR channel in secretion by duodenal villus enterocytes. Expression of CFTR in human and rat duodenum was characterized by immunofluorescence and immunoelectron microscopy by using anti-CFTR and enzyme marker antibodies. A subpopulation (2.5%) of villus enterocytes expressed very high levels of CFTR throughout the cells, with the greatest concentration apically. Thus CFTR-dependent export of HCO3 may be a property of villus as well as crypt cells. Finally, it was recently observed in clusters of rat and human duodenal enterocytes (218) that melatonin-induced [Ca2+]i signaling spread throughout the clusters, indicating that duodenal enterocytes interact and that the mucosa functions as a syncytium. This would enhance an integrated action of duodenal secretagogues.

A role for the CFTR channel in duodenal HCO3 export has been questioned on the basis of a study comparing transport of acid-base equivalents across the plasma membrane of proximal duodenal enterocytes in CFTR-deficient mice with that in normal littermates (188). The acid extrusion and the alkaline extrusion rates were unaffected by CFTR deficiency, and acid-base transport seemed mediated almost exclusively by Na+/H+ exchange, Cl/HCO3 exchange, and Na+-HCO3 cotransport. Absence of CFTR conductance in these studies might reflect the use of enterocytes rather than intact mucosa. The possibility cannot be excluded that cell isolation procedures induce cellular changes preventing migration of cytoplasmic CFTR (22, 251) to the duodenal enterocyte apical surface. The molecular expression and localization of Na+-HCO3 cotransporters (NBC) in mouse duodenum were recently studied using RT-PCR, sequence analysis, and immunohistochemistry (189). Enterocytes expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of duodenal villus enterocytes, whereas crypt enterocytes did not label with the anti-NBC antibodies. In membrane vesicles from rabbit duodenal enterocytes (120), pH gradient-driven Na+ uptake was partly HCO3 and DIDS sensitive and partly dependent on the Na+/H+ exchange isoform NHE1.

Na+/H+ exchange would supply intracellular HCO3 by export of H+ formed on hydration of CO2 to H+ and HCO3. The Na+/H+ exchanger isoforms NHE1 as well as NHE2 and NHE3 are expressed in mouse duodenal enterocytes, and all three isoforms were reported to contribute to regulation of intracellular pH (pHi) in these cells (187). In contrast, specific inhibition of the apical isoform NHE3 by the compound S3226 did not affect pHi in rat villus enterocytes in situ (85). However, the latter study (85) also showed that this compound causes a slowly developing rise in HCO3 secretion by duodenum in anesthetized rats. A rise in net alkaline (HCO3 minus H+) secretion could reflect inhibition of a smaller, and therefore concealed, H+ export. Luminal administration of the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid prevented the rise in net alkaline secretion, indicating an action on HCO3 export per se. The authors suggested interaction between NHE3 exchanger activity and the CFTR channel. Finally, entry of Cl for secretion across epithelial apical membranes predominantly depends on import of Cl by basolateral Na+-K+-2Cl cotransport (NKCC). Basal and cAMP-stimulated HCO3 secretion in duodenum from NKCC1 knockout mice was very similar to that in wild-type animals, indicating that apical export of HCO3 does not depend on basolateral Na+-K+-2Cl cotransport activity (254).


    STIMULATION OF GASTRODUODENAL BICARBONATE SECRETION BY LUMINAL ACID
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Presence of acid in the lumen is a powerful stimulant of gastric and duodenal HCO3 secretion. This has been demonstrated in several species, including humans (50, 116, 118), dogs (141), cats (67, 74), rats (72, 112, 216, 233), pigs (2), and frogs (99). The presence of a pH gradient within the mucus gel adherent to both gastric and duodenal mucosae raises the interesting question of how acid present in the lumen is sensed by the HCO3-secreting epithelium if the mucus pH gradient does indeed prevent a lowering of pH at the mucosal surface. An explanation for this is that the secreting mucosa senses PCO2 rather than pH. In support of this, CO2 generated at the surface during reaction between luminal H+ and secreted HCO3 has been shown to be the stimulus of alkaline secretion in rat in vivo (112). Acid and/or PCO2-sensitive cell filaments (221) or neural receptors protruding into the surface mucus gel could be an additional mechanism for sensing bulk luminal acidity.

The impaired alkaline response to acid in patients with duodenal ulcer disease (40, 108, 118, 163) has focused interest on mediation of this duodenal response. A low pH in the duodenal lumen (~pH 5 in rat and ~pH 3 in human) caused a marked, up to fivefold (60, 72) rise in the secretion. The response is mediated by neural reflexes and mucosal production of prostaglandins (23, 65, 235) and also, very likely, by locally produced uroguanylin (129). Several transmitters, including VIP and acetylcholine, are mediators of the efferent limb of the neural response. Chemical deafferentiation by capsaicin inhibits the rise in HCO3 secretion in response to luminal acid (114). The response to exogenous PGE2, in contrast, is not affected by the destruction of the afferent neurons. Thus this suggests that luminal acidification involves the enteric nervous system, whereas PGE2 acts directly on the HCO3-producing cells (23, 235). Recent findings suggest that prostaglandins stimulate duodenal HCO3 secretion by acting on duodenal EP3 receptors as well as EP4 receptors. Furthermore, it has been proposed (23) that stimulation via EP4 receptors is mediated by cAMP, whereas that via EP3 receptors is mediated by cAMP production as well as [Ca2+]i signaling. In addition, there is an interaction between these stimulatory pathways. In the stomach, prostaglandins stimulate HCO3 by affecting the gastric EP1 receptors (235). Affirming a role for prostaglandins and epithelial HCO3 in protection against mucosal injury, the duodenal mucosa in EP3 knockout mice has a markedly decreased ability to resist luminal acid damage (234). NO is another interesting mediator of the rise in duodenal alkaline secretion in response to luminal acid. Luminal acid stimulates the expression of inducible NO synthase in the duodenal villi, and it is proposed that this induces synthesis of NO (113).

It would seem physiologically rational that the presence of acid in the gastric lumen would result in an anticipatory rise in alkaline secretion by duodenal mucosa about to receive an acid load. However, instillation of acid into the ligated stomach or, conversely, decreasing gastric acidity by inhibition of acid secretion, does not affect duodenal HCO3 secretion (65, 70, 99).


    NEUROHUMORAL CONTROL OF BICARBONATE SECRETION
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
The stimulatory action of sham feeding demonstrates that gastric (80, 81, 141) and duodenal (26, 141) alkaline secretions are under central nervous system influence. Vagally mediated inhibition of duodenal HCO3 absorption also has been reported and explained by inhibition of villus Na+/H+ exchanger activity by an atropine-sensitive cholinergic mechanism (160). Studies in fasting rats demonstrated circadian rhythms in the gastric secretion of H+, HCO3, and mucus (147, 148). Interestingly, the secretion of HCO3 and that of H+ followed circadian rhythms with different peak times. This could, in theory, result in circadian rhythmicity of mucosal vulnerability to acid injury. Intracerebroventricular infusion of neurotransmitters and drugs has been used to further elucidate the central nervous system influence on gastroduodenal alkaline secretion. Centrally elicited stimulation of the duodenal secretion has been observed with some neuropeptides, including thyrotropin-releasing hormone (TRH), corticotropin-releasing factor (CRF), and bombesin (70, 151, 152), and with some benzodiazepines (199). Furthermore, the {alpha}1-receptor agonist phenylephrine (Fig. 4) caused a marked, up to fivefold, centrally elicited duodenal HCO3 secretion (149, 216, 217). Intrahypothalamic injection of CRF increased HCO3 secretion and also enhanced mucosal protection in rat stomach (95).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Proposed role of melatonin in control of duodenal mucosal HCO3 secretion. Melatonin released from intestinal enterochromaffin cells stimulates the protective secretion via an action at enterocyte MT2 receptors. Release is initiated by central nervous system (intracerebroventricular) administration of adrenoceptor agonist phenylephrine and by a low pH in the duodenal lumen. Neural pathways involve sympathetic as well as vagal components and the enteric nervous system (ENS). The action of melatonin at enterocyte membrane receptors also has been demonstrated in clusters of human duodenal enterocytes. [Adapted from Sjöblom et al. (216).]

 
A variety of peripherally acting agents have been found to influence HCO3 secretion in the duodenum and are summarized in Table 1. Primary sites of action include the enteric nervous systems, enterocyte membrane receptors, and local mucosal production of eicosanoids. Some of the more recently described secretagogues, namely, melatonin, uroguanylin, orexin A, and stress-related inhibitors of the duodenal secretion, are discussed below. Melatonin is released from the pineal gland in the central nervous system and from enterochromaffin cells in the intestinal epithelium. The amount produced in the intestine is ~400 times greater than that in the central nervous system, but the role of melatonin in gastrointestinal function has not been elucidated (39). Recent studies (215, 217) have demonstrated that close intraarterial infusion of melatonin and agonists into the duodenum in rats increased HCO3 secretion, with a low dose of melatonin (20 nmol·kg–1·h–1) causing maximal stimulation. Furthermore, melatonin induces enterocyte [Ca2+] signaling in clusters of human and rat duodenal enterocytes (218). The main pattern of response was similar to that observed in response to carbachol and cholecystokinin octapeptide (42) in duodenal enterocytes in primary culture. The responses to melatonin in vivo as well as on enterocyte signaling were inhibited by MT2-selective melatonin antagonists. Furthermore, the antagonist luzindole almost abolished the marked rise in secretion induced by intracerebroventricular infusion of the adrenoceptor agonist phenylephrine but did not affect the rise in release of melatonin to the duodenal lumen (216) induced by the central nervous system stimulation. This rise in secretion in response to central nervous system phenylephrine was also abolished by sublaryngeal ligation of all nerves around the carotid arteries but was unaffected by removal of either the pineal gland or pituitary gland. Melatonin thus stimulates duodenal HCO3 secretion via action at enterocyte MT2 receptors and mediates neural stimulation of the secretion. Interestingly, there is a strong disturbance of melatonin secretion in the exacerbation as well as remission stages of duodenal ulcer disease in patients (158).


View this table:
[in this window]
[in a new window]
 
Table 1. Peripherally acting transmitters and physiological conditions that affect duodenal mucosal bicarbonate secretion

 
Uroguanylin and guanylin are endogenous ligands for the transmembrane guanylate C receptor and increase enterocyte cGMP production. Both peptides are present throughout the length of the (rat) intestine. However, uroguanylin mRNA is most abundant in proximal and guanylin mRNA in distal segments of the small intestine, and uroguanylin, unlike guanylin, is resistant to digestion by the pancreatic enzyme chymotrypsin (84). Luminally applied uroguanylin (129) and guanylin (93, 192), like Escherichia coli heat-stable enterotoxin (Sta), stimulate HCO3 secretion by rat and mouse duodenal mucosa. The ratio between the HCO3 and total anion (Cl plus HCO3) secretory rates is considerably higher after stimulation with guanylin than after stimulation with a cAMP-dependent agonist such as PGE2 or VIP (93). Interestingly, acidification (pH 5.0–5.5) of the lumen of isolated mouse duodenum enhanced the stimulatory action of uroguanylin on mucosal HCO3 transport and short-circuit current (129). An important role of cGMP-dependent agonists in mucosal acid protection is further supported by the recent observation (192) that the rise in duodenal HCO3 secretion in response to luminal acid is markedly smaller in guanylate C receptor knockout mice than in wild-type animals.

Orexins are involved in the central nervous system’s control of appetite and behavior and appear to be involved in short-term regulation of feeding rather than long-term regulation of body weight (134, 145, 200). These peptides are present in endocrine cells and/or neurons in the intestine, but their effects on mucosal function and protection are largely unknown. Recent studies (77) have shown that close intraarterial infusion of low doses of orexin A (Fig. 5) caused a marked and dose-dependent stimulation of the duodenal HCO3 secretion in rats. However, stimulation occurred only in animals that had continuous access to their regular food. Short (overnight) deprivation of food abolished the stimulation of the duodenal HCO3 secretion. Similarly, short fasting caused a 100-fold increase in the amount of the muscarinic agonist bethanechol required for stimulation of the secretion. In contrast, the HCO3 secretory responses to VIP and melatonin were not affected (Fig. 5). An attractive explanation for the changes in sensitivity to orexin A and the muscarinic agonist would be that food constituents, either directly or indirectly by central or peripheral mechanisms, stimulate the activity or expression of signal pathways or receptors in the intestinal mucosa. It should also be noted that overnight fasting is a standard experimental procedure in studies of gastrointestinal function and pathophysiology in humans and animals. In view of these differences in response between the fed and fasted states, studies made on neuroendocrine control of mucosal protection and intestinal secretion may require reevaluation with respect to feeding status.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Short (overnight) food deprivation markedly decreases the sensibility of the HCO3 secreting rat duodenum to the appetite-regulating peptide orexin A. Similarly, short fasting caused a 100-fold increase in the amount of the muscarinic agonist bethanechol required for stimulation of the secretion. In contrast, the secretory responses to VIP and melatonin were not affected. Compounds were administered by close intraarterial infusion and HCO3 secretion continuously titrated. [Reprinted from Flemström et al. (77).]

 
It is likely that stress-induced reactions contribute to gastroduodenal damage. Splanchnicotomy or adrenergic blockade ameliorates stress-induced gastroduodenal ulceration in animals (65), whereas increased plasma levels of noradrenalin have been reported in patients with duodenal ulcer disease (123). The effects on mucosal HCO3 secretion resulting from elicitation of sympathetic reflexes and administration of {alpha}-adrenoceptor ligands and some neuropeptides have been studied in animals and humans (58, 128, 219). {alpha}2-Adrenoceptors (58, 62, 128, 139) as well as neuropeptide Y1 receptors (69) mediate sympathetic inhibition of mucosal HCO3 secretion and defense.


    DUODENAL PROTECTION AGAINST ACID
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
In the first part of the duodenum, mucosal HCO3 is secreted in immediate proximity to the epithelial cell surface and also proximal to the entry of HCO3 from pancreaticobiliary duct at the tubercle (papilla) of Vater. Despite this entry of pancreatic HCO3 distal to the duodenal bulb, luminal pH of the latter falls below pH 2.0 only sporadically and in short (5–10 s) spikes, and this is so even in humans with exocrine pancreatic secretory deficiency (177). Median pH in the duodenal bulb during the first hour after a meal was 3.99 in healthy controls and 3.79 in patients with pancreatic deficiency. In a recent study in fasting humans (122), gastric acid discharge and duodenal alkaline secretion were found to be dynamically coordinated with very few values of pH within the duodenal bulb being below pH 5.0. The juxtamucosal pH in rat duodenum in situ (72, 136) remained at neutrality during 15-min exposure to 10 mM HCl (pH 2.0) and decreased only slightly on exposure to 20 mM HCl. In healthy humans, the juxtamucosal pH in the duodenal bulb has been reported to remain at neutrality when luminal pH was as low as pH 1.5 (190). These combined data provide good evidence that in the healthy duodenum, the mucosa exports HCO3 at rates sufficient for maintaining its surface neutrality. In proximal duodenum, the adherent mucus layer is thinner than that in stomach, considerably so if one considers the firm mucus layer not removed by suction (Fig. 1). However, it would appear that, when necessary, it is of sufficient thickness to maintain a stable surface pH gradient under conditions of motility and low luminal pH in vivo.

Studies in several species with a variety of modulators of secretion demonstrate the key importance of epithelial HCO3 secretion in duodenal mucosal protection against luminal acid. Stimulation and inhibition of alkaline secretion respectively increase and decrease the juxtamucosal pH at the duodenal mucosal cell surface (72, 136). Stimulation of mucosal HCO3 secretion by VIP, at doses not affecting mucosal blood flow, protected the duodenal mucosa in rat (175) and pig (1) against morphological damage induced by 10 mM luminal HCl and 30 mM luminal HCl, respectively. Stimulation of mucosal HCO3 secretion by glucagon significantly reduced the duodenal mucosal damage induced by 50 mM HCl in the rabbit (258). Inhibition of mucosal HCO3 secretion by parenteral administration of NH4Cl, vasopressin, or furosemide, in contrast, increased duodenal mucosal damage (258). Modest, acute hypovolemia markedly inhibited mucosal alkaline secretion in rat duodenum (125, 128). This procedure also significantly increased the morphological changes induced by 15-min exposure of the duodenal lumen to 100 mM HCl (125), although it had no effect at the lower concentration (10 mM) of acid. Furthermore, E-type prostaglandins, which increase duodenal mucosal alkaline secretion in all species tested, in vivo as well as in vitro, also increase mucosal resistance to luminal acid (64, 116, 233, 234, 235, 263). Inhibition of mucosal endogenous prostaglandin production by COX inhibitors reduces the alkaline secretion in humans (162) and in several (2, 64, 72, 233, 235) but not all (172) experimental models, and these compounds are well-known inducers of duodenal ulceration in experimental animals as well as in humans (229, 256).

An acid-protective role for the duodenal mucosal alkaline secretion is further supported by the finding that HCO3 secretion in proximal duodenum (40, 108, 118, 163), and, in particular, the rise in secretion in response to luminal acid is depressed in patients with duodenal ulcer disease. It has also been reported the pH at the surface of this epithelium in duodenal ulcer patients is lower than that in healthy subjects (190). The HCO3 secretion is reported to normalize after eradication of the bacterium Helicobacter pylori in patients with such infection (108). Interestingly, evidence has been presented that H. pylori infection inhibits antral mucosal production of NO (253), a transmitter thought to be important in mediation of the duodenal alkaline response (113). Perhaps surprisingly, E-type prostaglandin release from duodenal mucosa in patients with duodenal ulcer disease was greater than that in healthy controls (40). This could indicate that decreased sensitivity to E-type prostaglandins also may be part of the depression of the alkaline secretion in duodenal ulcer disease.

One additional mechanism of duodenal defense, namely, intracellular neutralization of acid, has been proposed from studies of pHi in apical villus cells in rat duodenum in situ. pHi was measured using pH-sensitive microelectrodes (178) or fluorescence microscopy of the duodenal surface after loading with the pH-sensitive compound BCECF-AM (4, 5). During acid perfusion, pHi decreased, whereas mucus gel thickness and blood flow increased. At the highest luminal acidity tested (pH 2.2), the recorded decrease in pHi after 5 min was from ~7.10 to ~6.30 with the fluorescence microscopy technique. The changes in pHi recorded with intracellular microelectrodes, and in response to a somewhat higher acidity (10 mM HC), were smaller (from pH 7.46 to pH 7.21) than those recorded with fluorescence microscopy. With both techniques, this decrease in pHi was smaller on a second brief exposure to acid. This interesting adaptation to rapid shifts in duodenal luminal acidity was sensitive to DIDS and was explained by acid-induced augmented cellular uptake of HCO3 by the enterocyte basolateral Na+-HCO3 cotransporter. Subsequent perfusion with neutral luminal solution increased pHi above baseline values (4), and, in line with the higher rates of transcellular HCO3 transport in proximal duodenum (115, 116, 214), acid-induced decreases in pHi were smaller in proximal than in distal duodenum (178). It thus would seem likely that intracellular neutralization of acid reflects the basolateral part of the processes for transcellular transport of HCO3 into the duodenal lumen (Fig. 3). It should be noted that, mainly, pHi in apical enterocytes in the duodenal villi was examined in these studies and that decreases in pHi may reflect acidification by high levels of CO2 (98, 112, 122) formed within the duodenal mucus gel during reaction between acid from the lumen and secreted HCO3.


    CONCLUSIONS
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Since the first experimental evidence for the mucus bicarbonate barrier was reported nearly three decades ago, it has become firmly established as a key component of the gastroduodenal mucosal protective mechanisms against acid and pepsin. The secretion of HCO3 into a stable, adherent mucus gel layer creates a pH gradient at the epithelial surface in stomach and duodenum and provides the first line of mucosal defense against luminal acid. The mucus gel layer is an effective barrier to luminal pepsin, and evidence points to it as the major protective mechanism against proteolytic digestion of the underlying epithelium by this enzyme.

The numerous pH gradient studies are good experimental evidence for the existence of the mucus bicarbonate barrier in vivo and the establishment of a nearly neutral pH at the epithelial surface. The gastric epithelial cells may be slightly and transiently acidified during acute exposure to high concentrations of luminal acid, but mainly, the surface epithelium in acid-secreting gastric mucosa exports HCO3 at rates sufficient to maintain a neutral pH at the apical cell surfaces. During acid secretion, parietal cells release HCO3 into the mucosal interstitium and vasculature. This increased interstitial HCO3, the alkaline tide, is imported by the surface epithelial cells, significantly enhancing mucosal alkaline secretion and surface alkalinity. Uncertainties have been raised as to whether a more alkaline or acidic juxtamucosal pH exists at the mucosal surface at higher luminal pH values (pH 5), and more experimentation is needed to resolve this question. The two approaches to studying pH gradients are not directly comparable either, because pH-sensitive microelectrode studies, at luminal pH 1–3, have been conducted primarily in unstirred conditions, whereas the confocal imaging studies at pH 3–5 were performed in a perfused system.

In duodenum, the mucosal HCO3 secretion is currently accepted as the primary defense mechanism against acid discharge from the stomach. Thus the duodenal epithelium secretes HCO3 at higher rates (per unit surface area) than does the stomach or, for that matter, the more distal small intestine. In the first part of the duodenum, mucosal HCO3, secreted proximal to the entry of HCO3 from the pancreaticobiliary duct, maintains a pH gradient with a neutral pH at the mucus epithelial interface under all luminal acidities encountered in the healthy organ. Presence of acid in the lumen is a powerful stimulant in duodenum and causes up to a fivefold increase in duodenal HCO3 secretion as well as an increased mucus gel secretion. Furthermore, there is an impaired HCO3 secretory response to acid in patients with duodenal ulcer disease. The alkaline response to acid is mediated by neural reflexes and mucosal production of prostaglandins and, as shown recently, by locally produced uroguanylin. Melatonin released from enterochromaffin cells in the duodenal mucosa, furthermore, may be involved in control of HCO3 secretion, and a role also has been proposed for local mucosal dopamine.

Finally, this review is confined to aspects of the mucus bicarbonate barrier and this barrier’s role in protection against the natural endogenous aggressors acid and pepsin. Although there is an array of other aggressive factors that have been shown to be integral to the pathology of peptic ulceration, the principle as proposed by Schwartz in 1910 (208) still holds: no peptic activity, no ulcer. Furthermore, it should be stressed that the mucus bicarbonate barrier is one of many integrated components that form the complete protective mucosal barrier. It is the initial, and only, preepithelial barrier between the lumen and the gastric surface epithelium, and as discussed in this review, evidence suggests that it is sufficient for protection against luminal acid and pepsin during normal digestive processes. Mucosal protection for parietal and chief cells within the gastric crypts is different, with their cell apical membranes apparently resistant to their intracryptal secretions (37, 245). When the mucus bicarbonate barrier is overwhelmed or when it breaks down in disease, then there are a whole series of protective mechanisms that come into play, including intracellular neutralization of acid, rapid epithelial repair and maintenance, and distribution of mucosal blood flow.


    GRANTS
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
This work was supported by Swedish Research Council Grant 3515 and the Wallenberg Foundation (to G. Flemström).


    ACKNOWLEDGMENTS
 
We thank Dr. Markus Sjöblom and Dr. Olof Nylander for discussions and comments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Flemström, Division of Physiology, Dept. of Neuroscience, Uppsala Univ., BMC, PO Box 572, SE-751 23 Uppsala, Sweden (E-mail: Gunnar.Flemstrom{at}Fysiologi.uu.se)


    REFERENCES
 TOP
 ABSTRACT
 ADHERENT MUCUS GEL LAYER...
 STRUCTURE AND STABILITY OF...
 TREFOIL PEPTIDES AND THE...
 FACTORS INFLUENCING MUCUS LAYER...
 MUCUS AND PROTECTION AGAINST...
 pH GRADIENT AT THE...
 ROLE OF THE ALKALINE...
 SECRETION OF ACID AND...
 GASTRIC BICARBONATE SECRETION
 DUODENAL ALKALINE SECRETION
 STIMULATION OF GASTRODUODENAL...
 NEUROHUMORAL CONTROL OF...
 DUODENAL PROTECTION AGAINST ACID
 CONCLUSIONS
 GRANTS
 REFERENCES
 
1. Ainsworth MA, Fenger C, Svendsen P, and Schaffalitzky de Muckadell OB. Effect of stimulation of mucosal HCO3 secretion on acid-induced injury to porcine duodenal mucosa. Scand J Gastroenterol 28: 1091–1097, 1993.[ISI][Medline]

2. Ainsworth MA, Svendsen P, Glad H, Andersen NJ, Olsen O, and Schaffalitzky de Muckadell OB. Duodenal mucosal bicarbonate secretion in pigs is accompanied by compensatory changes in pancreatic and biliary HCO3 secretion. Scand J Gastroenterol 29: 889–896, 1994.[ISI][Medline]

3. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, and Kaunitz JD. Sensory pathways and cyclooxygenase regulate mucus gel thickness in rat duodenum. Am J Physiol Gastrointest Liver Physiol 280: G470–G474, 2001.[Abstract/Free Full Text]

4. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, and Kaunitz JD. Acute adaptive cellular base uptake in rat duodenal epithelium. Am J Physiol Gastrointest Liver Physiol 280: G1083–G1092, 2001.[Abstract/Free Full Text]

5. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, Sassani P, Dukkipatis R, Pushkin A, Kurtz I, and Kaunitz JD. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury. J Clin Invest 108: 1807–1816, 2001.[Abstract/Free Full Text]

6. Akiba Y, Guth PH, Engel E, Nastaskin I, and Kaunitz JD. Dynamic regulation of mucus thickness in the rat duodenum. Am J Physiol Gastrointest Liver Physiol 279: G437–G447, 2000.[Abstract/Free Full Text]

7. Allen A. Structure of gastrointestinal mucus and the viscous and gel-forming properties of mucus. Br Med Bull 34: 28–33, 1978.[Medline]

8. Allen A. Gastrointestinal mucus. In: Handbook of Physiology. Gastrointestinal Physiology. Salivary, Gastric, Pancreatic, and Hepatobiliary Secretion. Bethesda, MD: Am. Physiol. Soc. 1989, sect. 6, vol. III, chapt. 19, p. 359–382.

9. Allen A and Carroll NJH. Adherent and soluble mucus in the stomach and duodenum. Dig Dis Sci 30: 55S–62S, 1985.[Medline]

10. Allen A, Cunliffe WF, Pearson JP, and Venerables CW. The adherent gastric mucus gel barrier in man and changes in peptic ulceration. J Intern Med 228, Suppl 732: 83–90, 1990.[Medline]

11. Allen A, Flemström L, Garner A, and Kivilaakso E. Gastroduodenal mucosal protection. Physiol Rev 73: 823–857, 1993.[Abstract/Free Full Text]

12. Allen A, Flemström G, Garner A, Silen W, and Turnberg L. Mechanisms of Mucosal Protection in the Upper Gastrointestinal Tract. New York: Raven, 1984.

13. Allen A and Garner A. Gastric mucus and bicarbonate secretion and their possible role in mucosal protection. Gut 21: 249–262, 1980.[ISI][Medline]

14. Allen A, Hutton DA, Leonard AJ, Pearson JP, and Sellers LA. The role of mucus in the protection of the gastroduodenal mucosa. Scand J Gastroenterol 21, Suppl 125: 71–77, 1986.[ISI]

15. Allen A, Hutton D, McQueen S, Garner A. Dimensions of the gastroduodenal surface pH gradients exceed those of the adherent mucus gel layer. Gastroenterology 85: 463–476, 1983.[ISI][Medline]

16. Allen A, Leonard AJ, Pearson JP, Sellers LAA, and Bennet MA. Pepsin induced mucosal damage compared with that with ethanol or hypertonic saline: the role of the adherent mucus barrier. In: Mechanisms of Injury and Mucosal Repair in the Upper Gastrointestinal Tract, edited by Garner A and O'Brien, P. New York: Wiley, 1991, p. 7–18.

17. Allen A, Pain RH, and Robson T. Model for the structure of gastric mucus gel. Nature 264: 88–89, 1976.[ISI][Medline]

18. Allen A and Pearson JP. The gastrointestinal adherent mucous gel barrier. In: Glycoprotein Methods and Protocols: The Mucins, edited by Corfield AP. Totowa, NJ: Humana, 2000, p. 57–64.

19. Allen A and Snary D. The structure and function of gastric mucus. Gut 13: 666–672, 1972.[ISI][Medline]

20. Altamirano M, Requena M, and Perez TZ. Interstitial fluid pressure and gastric alkaline secretion. Am J Physiol 229: 1421–1426, 1975.[Abstract/Free Full Text]

21. Ameen NA, Ardito T, Kashgarian M, and Marino CR. A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator. Gastroenterology 108: 1016–1023, 1995.[ISI][Medline]

22. Ameen NA, Mårtensson B, Bourguinon L, Marino C, Isenberg J, and McLaughlin GE. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo. J Cell Sci 112: 887–894, 1999.[Abstract/Free Full Text]

23. Aoi M, Aihara E, Nakashima M, and Takeuchi K. Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats. Am J Physiol Gastrointest Liver Physiol 287: G96–G103, 2004.[Abstract/Free Full Text]

24. Atuma C, Strugala V, Allen A, and Holm L. The adherent gastric mucus gel layer: thickness and physical state in vivo. Am J Physiol Gastrointest Liver Physiol 280: G922–G929, 2001.[Abstract/Free Full Text]

25. Bahari H, Ross IN, and Turnberg LA. Demonstration of a pH gradient across the mucus layer on the surface of human gastric mucosa in vitro. Gut 23: 513–516, 1982.[Abstract]

26. Ballesteros MA, Wolosin JD, Hogan DL, Koss MA, and Isenberg JI. Cholinergic regulation of human proximal duodenal mucosal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 261: G327–G331, 1991.[Abstract/Free Full Text]

27. Bansil R, Stanley E, and Lamont JT. Mucin biophysics. Annu Rev Physiol 57: 635–657, 1995.[CrossRef][ISI][Medline]

28. Baumgartner HK, Kirbiyik U, Coskun T, Chu S, and Montrose MH. Endogenous cyclooxygenase activity regulates mouse gastric surface pH. J Physiol 544: 871–882, 2002.[Abstract/Free Full Text]

29. Baumgartner HK and Montrose MH. Regulated alkali secretion acts in tandem with unstirred layers to regulate mouse gastric surface pH. Gastroenterology 126: 774–783, 2004.[CrossRef][ISI][Medline]

30. Bell A, Allen A, Morris ER, and Ross-Murphy SB. Functional interactions of gastric mucus glycoprotein. Int J Biol Macromol 6: 309–315, 1984.[CrossRef][ISI]

31. Bell A, Sellers LA, Allen A, Cunliffe WJ, Morris ER, and Ross-Murphy SB. Properties of gastric and duodenal mucus: effect of proteolysis, disulfide reduction, bile, acid, ethanol and hypertonicity on mucus gel structure. Gastroenterology 88: 269–280, 1985.[ISI][Medline]

32. Bertrand CA and Frizzell RA. The role of regulated CFTR trafficking in epithelial secretion. Am J Physiol Cell Physiol 285: C1–C18, 2003.[Abstract/Free Full Text]

33. Bhaskar K, Garik P, Turner BS, Bradley JD, Bansil R, Stanley HE, and Lamont JT. Viscous fingering of HCl through gastric mucin. Nature 360: 458–461, 1992.[CrossRef][ISI][Medline]

34. Bhaskar K, Gong D, Bansil R, Pajevic S, Hamilton JA, Turner BS, and Lamont JT. Profound increase in viscosity and aggregation of pig gastric mucin at low pH. Am J Physiol Gastrointest Liver Physiol 261: G827–G833, 1991.[Abstract/Free Full Text]

35. Biasco G, Paganelli GM, Vaira D, Holton J, Di Febo G, Brillanti S, Miglioli L, Barbara L, and Samloff MI. Serum pepsinogen 1 and 11 concentrations and IgG antibody to Helicobacter pylori in dyspeptic patients. J Clin Pathol 46: 826–828, 1993.[Abstract]

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

37. Boron WF, Waisbren SJ, Modlin IM, and Geibel JP. Unique permeability barrier of the apical surface of parietal and chief cells in isolated perfused gastric glands. J Exp Biol 196: 347–360, 1994.[Abstract/Free Full Text]

38. Brockhausen I and Schacter H. Glycosyltransferases involved in N- and O-glycan biosynthesis. Glycosciences, Status and Perspectives, edited by Gabius H-J and Gabius S. London: Chapman, 1997, p. 1–14.

39. Bubenik GA. Gastrointestinal melatonin: localization, function, and clinical relevance. Dig Dis Sci 47: 2336–2348, 2002.[CrossRef][ISI][Medline]

40. Bukhave K, Rask-Madsen J, Hogan DL, Koss MA, and Isenberg JI. Proximal duodenal prostaglandin E2 release and mucosal bicarbonate secretion are altered in patients with duodenal ulcer. Gastroenterology 99: 951–955, 1990.[ISI][Medline]

41. Cao X, Bansil R, Bhaskar KR, Turner BS, LaMont JT, Niu N, and Afdhal NH. pH-dependent conformational change of gastric mucin leads to sol-gel transition. Biophys J 76: 1250–1258, 1999.[Abstract/Free Full Text]

42. Chew CS, Säfsten B, and Flemström G. Calcium signaling in cultured human and rat duodenal enterocytes. Am J Physiol Gastrointest Liver Physiol 275: G296–G304, 1998.[Abstract/Free Full Text]

43. Chikhlssa AR, Gharzouli A, Charpin G, Descroix-Vagne M, and Pansu D. Comparison of VIP-induced electrolyte secretion in rat small intestine. Reprod Nutr Dev 32: 37–45, 1992.[ISI][Medline]

44. Chu S, Tanaka S, Kaunitz J, and Montrose MH. Dynamic regulation of gastric surface pH by luminal pH. J Clin Invest 103: 605–612, 1999.[Abstract/Free Full Text]

45. Clarke LL and Harline MC. Dual role of CFTR in cAMP-stimulated HCO3 secretion across murine duodenum. Am J Physiol Gastrointest Liver Physiol 274: G718–G726, 1998.[Abstract/Free Full Text]

46. Corfield A, Carroll D, Myerscough N, and Probert SJ. Mucus in the gastrointestinal tract in health and disease. Front Biosci 6: D1321–D1357, 2001.[ISI][Medline]

47. Corfield A, Myerscough N, Myerscough N, Longman R, Sylvester P, Arul S, and Pignatelli M. Mucins and mucosal protection in the gastrointestinal tract: new prospects or mucins in the pathology of gastrointestinal disease. Gut 47: 589–594, 2000.[Free Full Text]

48. Coskun T, Baumgartner HK, Chu S, and Montrose MH. Coordinated regulation of gastric chloride secretion with both acid and alkali secretion. Am J Physiol Gastrointest Liver Physiol 283: G1147–G1155, 2002.[Abstract/Free Full Text]

49. Coskun T, Chu S, and Montrose MH. Intragastric pH regulates conversion from net acid to net alkaline secretion by the rat stomach. Am J Physiol Gastrointest Liver Physiol 281: G870–G877, 2001.[Abstract/Free Full Text]

50. Crampton JR, Gibbons LC, and Rees WDW. Effect of luminal pH on the output of bicarbonate and PGE2 by normal human stomach. Gut 28: 1291–1295, 1987.[Abstract]

51. Cummings GM, Grossman MI, and Ivy AC. An experimental study of the acid factor in ulceration of the gastrointestinal tract in dogs. Gastroenterology 10: 714–726, 1948.[ISI]

52. Davenport HW. Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50: 487–499, 1966.[ISI][Medline]

53. Debellis L, Caroppo R, Frömter E, and Curci S. Alkaline secretion by frog gastric glands measured with pH microelectrodes in the gland lumen. J Physiol 513: 235–241, 1998.[Abstract/Free Full Text]

54. De Bolos C, Francisco XR, and Lopez-Ferrer A. Regulation of mucin and glycoconjugate expression: from normal epithelium to gastric tumors. Front Biosci 6: D1256–D1263, 2001.[ISI][Medline]

55. Desai M, Mutlu M, and Vadgama P. A study of macromolecular diffusion through native porcine mucus. Experientia 48: 22–26, 1992.[ISI][Medline]

56. Dixon J, Strugala V, Griffin SM, Welfare MR, Dettmar PW, Allen A, and Pearson JP. Eosophageal mucin: an adherent mucus gel barrier is absent in the normal eosophagus but present in columnar-lined Barrett's esophagus. Am J Gastroenterol 96: 2575–2583, 2001.[CrossRef][ISI][Medline]

57. Engel E, Guth PH, Nishizaki Y, and Kaunitz JD. Barrier function of the gastric mucus gel. Am J Physiol Gastrointest Liver Physiol 269: G994–G999, 1995.[Abstract/Free Full Text]

58. Fändriks L, Jönson C, and Nylander O. Effects of splanchnic nerve stimulation and of clonidine on gastric and duodenal HCO3 secretion in the anaesthetized cat. Acta Physiol Scand 130: 251–258, 1987.[ISI][Medline]

59. Farrell J, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DK, and Wang TC. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J Clin Invest 109: 193–204, 2002.[Abstract/Free Full Text]

60. Feitelberg SP, Hogan DL, Koss MA, and Isenberg JI. pH threshold for human duodenal bicarbonate secretion and diffusion of CO2. Gastroenterology 102: 1252–1258, 1992.[ISI][Medline]

61. Flemström G. Active alkalinization by amphibian gastric fundic mucosa. Am J Physiol Endocrinol Metab Gastrointest Physiol 233: E1–E12, 1977.[Abstract/Free Full Text]

62. Flemström G. Effect of catecholamines, Ca++ and gastrin on gastric HCO3 secretion. In: Gastric Ion Transport: Proceedings from the Symposium on Gastric Ion Transport in Uppsala, Sweden, July 24–28, 1977, edited by Öbrink KJ and Flemström G. Uppsala, Sweden: Acta Physiologica Scandinavica, 1978, p. 81–90.

63. Flemström G. Cl dependence of HCO3 transport in frog gastric mucosa. Ups J Med Sci 85: 303–309, 1980.[ISI][Medline]

64. Flemström G. Stimulation of bicarbonate transport in isolated bullfrog duodenum by prostaglandins. Am J Physiol Gastrointest Liver Physiol 239: G198–G203, 1980.[Abstract/Free Full Text]

65. Flemström G. Gastric and duodenal mucosal bicarbonate secretion. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR, Jacobson ED, Christensen J, Alpers D, and Walsh JH. New York: Raven, 1994, p. 1285–1309.

66. Flemström G and Garner A. Gastroduodenal bicarbonate transport: characteristics and proposed role in acidity regulation and mucosal protection. Am J Physiol Gastrointest Liver Physiol 242: G183–G193, 1982.[Abstract/Free Full Text]

67. Flemström G, Garner A, Nylander O, Hurst BC, and Heylings JR. Surface epithelial bicarbonate transport by mammalian duodenum in vivo. Am J Physiol Gastrointest Liver Physiol 243: G348–G358, 1982.[Abstract/Free Full Text]

68. Flemström G, Hällgren A, Nylander O, Engstrand L, Wilander E, and Allen A. Adherent surface mucus gel restricts diffusion of macromolecules in rat duodenum in vivo. Am J Physiol Gastrointest Liver Physiol 277: G375–G382, 1999.[Abstract/Free Full Text]

69. Flemström G and Isenberg JI. Gastroduodenal mucosal alkaline secretion and mucosal protection. News Physiol Sci 16: 23–28, 2001.[ISI][Medline]

70. Flemström G and Jedstedt G. Stimulation of duodenal mucosal bicarbonate secretion in the rat by brain peptides. Gastroenterology 97: 412–420, 1989.[ISI][Medline]

71. Flemström G, Jedstedt G, and Nylander O. {beta}-Endorphin and enkephalins stimulate duodenal mucosal alkaline secretion in the rat in vivo. Gastroenterology 90: 368–372, 1986.[ISI][Medline]

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

73. Flemström G, Kivilaakso E, Bridén S, Nylander O, and Jedstedt G. Gastroduodenal bicarbonate secretion in mucosal protection. Possible role of vasoactive intestinal peptide and opiates. Dig Dis Sci 30: 63S–68S, 1985.[Medline]

74. Flemström G and Nylander O. Stimulation of duodenal epithelial HCO3 transport in the guinea pig and cat by luminal prostaglandin E2. Prostaglandins 21, Suppl: 47–52, 1981.[CrossRef][Medline]

75. Flemström G and Sachs G. Properties of isolated antral mucosa. I. General characteristics. Am J Physiol 228: 1188–1198, 1975.[Abstract/Free Full Text]

76. Flemström G, Säfsten B, and Jedstedt G. Stimulation of mucosal alkaline secretion in rat duodenum by dopamine and dopaminergic compounds. Gastroenterology 104: 825–833, 1993.[ISI][Medline]

77. Flemström G, Sjöblom M, Jedstedt G, and Åkerman KEO. Short fasting dramatically decreases rat duodenal secretory responsiveness to orexin-A but not to VIP or melatonin. Am J Physiol Gastrointest Liver Physiol 285: G1091–G1096, 2003.[Abstract/Free Full Text]

78. Flemström G and Turnberg LA. Gastroduodenal defense mechanisms. Clin Gastroenterol 13: 327–354, 1984.[ISI][Medline]

79. Florey H. Mucin and protection of the body. Proc R Soc Lond B Biol Sci 143: 144–148, 1955.

80. Forssell H, Lind T, and Olbe L. Comparative potency of carbachol, sham feeding, fundic distension and 16,16-dimethyl prostaglandin E2 in stimulating human gastric bicarbonate secretion. Acta Physiol Scand 134: 75–78, 1988.[ISI][Medline]

81. Forssell H, Stenquist B, and Olbe L. Vagal stimulation of human gastric bicarbonate secretion. Gastroenterology 89: 581–586, 1985.[ISI][Medline]

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

83. Forstner JF and Forstner G. Gastrointestinal mucus. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR, Jacobson ED, Christensen J, Alpers D, and Walsh JH. New York: Raven, 1994, p. 1255–1283.

84. Forte LR. Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology. Regul Pept 81: 25–39, 1999.[CrossRef][ISI][Medline]

85. Furukawa O, Bi LC, Guth PH, Engel E, Hirokawa M, and Kaunitz JD. NHE3 inhibition activates duodenal bicarbonate secretion in the rat. Am J Physiol Gastrointest Liver Physiol 286: G102–G109, 2004.[Abstract/Free Full Text]

86. Gannon B, Browning J, O'Brien P, and Rogers P. Mucosal microvascular architecture of the fundus and body of the human stomach. Gastroenterology 86: 866–875, 1984.[ISI][Medline]

87. Garner A and Flemström G. Gastric bicarbonate secretion in the guinea pig. Am J Physiol Endocrinol Metab Gastrointest Physiol 234: E535–E542, 1978.[Abstract/Free Full Text]

88. Gendler S and Spicer AP. Epithelial mucin genes. Annu Rev Physiol 57: 607–634, 1995.[CrossRef][ISI][Medline]

89. Glad H, Ainsworth MA, Svendsen P, Fahrenkrug J, and Schaffalitzky de Muckadell OB. Effect of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide on pancreatic, hepatic and duodenal mucosal bicarbonate secretion in the pig. Digestion 67: 56–66, 2003.[CrossRef][ISI][Medline]

90. Goddard P, Kao YCJ, and Lichtenburger L. Luminal surface hydrophobicity of canine gastric mucosa is dependent on surface mucous gel. Gastroenterology 98: 361–370, 1990.[ISI][Medline]

91. Godl K, Johansson MEV, Lidell ME, Morgelin M, Karlsson H, Olson FJ, Gum JR, Kim YS, and Hansson GC. The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment. J Biol Chem 277: 47248–47256, 2002.[Abstract/Free Full Text]

92. Gouyer V, Wiede A, Buisine MP, Dekeyser S, Moreau O, Lesuffleur T, Hoffman W, and Huet G. Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype. Biochim Biophys Acta 1539: 71–84, 2001.[CrossRef][ISI][Medline]

93. Guba M, Kuhn M, Forssmann WG, Classen M, Gregor M, and Seidler U. Guanylin strongly stimulates rat duodenal HCO3 secretion: proposed mechanism and comparison with other secretagogues. Gastroenterology 111: 1760–1763, 1996.[ISI][Medline]

94. Guha S and Kaunitz JD. Gastroduodenal mucosal defense: an integrated protective response. Curr Opin Gastroenterol 18: 650–657, 2002.[CrossRef][ISI]

95. Gunion MW, Kauffman GI, and Taché Y. Intrahypothalamic corticotropin-releasing factor elevates gastric bicarbonate secretion and inhibits stress ulcers in rats. Am J Physiol Gastrointest Liver Physiol 258: G152–G157, 1990.[Abstract/Free Full Text]

96. Hällgren A, Flemström G, Hellström PM, Lördal MS, Hellgren S, and Nylander O. Neurokinin A increases duodenal mucosal permeability, bicarbonate secretion, and fluid output in the rat. Am J Physiol Gastrointest Liver Physiol 273: G1077–G1086, 1997.[Abstract/Free Full Text]

97. Hällgren A, Flemström G, and Nylander O. Interaction between Neurokinin A, VIP, prostanoids and enteric nerves in regulation of duodenal function. Am J Physiol Gastrointest Liver Physiol 275: G95–G103, 1998.[Abstract/Free Full Text]

98. Harmon JW, Woods M, and Gurll NJ. Different mechanisms of hydrogen ion removal in stomach and duodenum. Am J Physiol Endocrinol Metab Gastrointest Physiol 235: E692–E698, 1978.[Free Full Text]

99. Heylings JR, Garner A, and Flemström G. Stimulation of gastroduodenal HCO3 transport by luminal acid in the frog in vitro. Am J Physiol Gastrointest Liver Physiol 246: G235–G242, 1984.[Abstract/Free Full Text]

100. Hidaka E, Ota H, Hidaka H, Hayama M, Matsuzawa K, Akamatsu T, Nakayama J, and Katsuyama T. Helicobacter pylori and two ultrastructurally distinct layers of gastric mucous cell mucins in the surface mucous gel layer. Gut 49: 474–480, 2001.[Abstract/Free Full Text]

101. Hills BA. Gastric mucosal barrier stabilisation of the hydrophobic lining to the stomach by mucus. Am J Physiol Gastrointest Liver Physiol 249: G342–G349, 1985.[Abstract/Free Full Text]

102. Hirschowitz B. Pepsin and the esophagus. Yale J Biol Med 72: 133–143, 1999.[ISI][Medline]

103. Ho S, Anway RE, and Khalil ZM. Mucin gene expression in normal and neoplastic tissue and use of mucin mRNAs as markers for micrometastatic disease. J Clin Ligand Assay 22: 358–363, 1999.[ISI]

104. Ho S, Anway RE, and Khalil ZM. Spatial organization of MUC5AC and MUC6 mucins within the surface mucous layer of the stomach (Abstract). Gastroenterology 118: 1396, 2000.

105. Ho S, Roberton 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]

106. Hoffmann WJW and Wiede A. Molecular medicine of TFF-peptides: from gut to brain. Histol Histopathol 16: 319–334, 2001.[ISI][Medline]

107. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky de Muckadell OB, and Ainsworth MA. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO3 secretion. Am J Physiol Gastrointest Liver Physiol 272: G872–G878, 1997.[Abstract/Free Full Text]

108. Hogan Rapier RC DL, Dreilinger A, Koss MA, Basuk PM, Weinstein WM, Nyberg LM, and Isenberg JI. Duodenal bicarbonate secretion: eradication of Helicobacter pylori and duodenal structure and function in humans. Gastroenterology 110: 705–716, 1996.[ISI][Medline]

109. Hollander F. The two-component mucus barrier; its activity in protecting the gastroduodenal mucosa against peptic ulceration. AMA Arch Intern Med 93: 107–120, 1954.

110. Holm L, Ågren J, and Persson AEG. Stimulation of acid secretion increases the gastric luminal pressure in the rat. Gastroenterology 103: 1797–1803, 1992.[ISI][Medline]

111. Holm L and Flemström G. Microscopy of acid transport at the gastric surface in vivo. J Intern Med 228, Suppl 732: 91–95, 1990.[ISI][Medline]

112. Holm M, Johansson B, Pettersson A, and Fändriks L. Carbon dioxide mediates duodenal mucosal alkaline secretion in response to luminal acidity in the anesthetized rat. Gastroenterology 115: 680–685, 1998.[ISI][Medline]

113. Holm M, Powell T, Casselbrant A, Johansson B, Fändriks L. Dynamic involvement of the inducible type of nitric oxide synthase in acid-induced duodenal mucosal alkaline secretion in the rat. Dig Dis Sci 46: 1765–1771, 2001.[CrossRef][ISI][Medline]

114. Holzer P. Gastroduodenal mucosal defense: coordination by a network of messengers and mediators. Curr Opin Gastroenterol 17: 489–496, 2001.[CrossRef][ISI]

115. Isenberg JI, Flemström G, and Johansson C. Mucosal bicarbonate secretion is significantly greater in the proximal versus distal duodenum in the in vivo rat. In: Mechanisms of Mucosal Protection in the Upper Gastrointestinal Tract, edited by Allen A, Flemström G, Garner A, Silen W, and Turnberg LA. New York: Raven, 1984, p. 175–180.

116. Isenberg JI, Hogan DL, Koss MA, and Selling JA. Human duodenal mucosal bicarbonate secretion. Evidence for basal secretion and stimulation by hydrochloric acid and a synthetic prostaglandin E1 analogue. Gastroenterology 91: 370–378, 1986.[ISI][Medline]

117. Isenberg JI, Ljungström M, Säfsten B, and Flemström G. Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl-HCO3 exchange and NaHCO3 cotransport. Am J Physiol Gastrointest Liver Physiol 265: G677–G685, 1993.[Abstract/Free Full Text]

118. Isenberg JI, Selling JA, Hogan DL, and Koss MA. Impaired proximal duodenal mucosal bicarbonate secretion in duodenal ulcer patients. N Engl J Med 316: 374–379, 1987.[Abstract]

119. Ito S, Lacy ER, Rutten MJ, Critchlow J, and Silen W. Rapid repair of injured gastric mucosa. Scand J Gastroenterol Suppl 101: 87–95, 1984.[Medline]

120. Jacob P, Christiani S, Rossmann H, Lamprecht G, Vieillard-Baron D, Müller R, Gregor M, Seidler U. Role of Na+HCO3 cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion. Gastroenterology 119:406–419, 2000.[ISI][Medline]

121. Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu E, Gregor M, Groneberg DA, Kere J, and Seidler U. Down-regulated in adenoma mediates apical Cl/HCO3 exchange in rabbit, rat, and human duodenum. Gastroenterology 122: 709–724, 2002.[ISI][Medline]

122. Järbur K, Dalenbäck J, and Sjövall H. Quantitative assessment of motility-associated changes in gastric and duodenal luminal pH in humans. Scand J Gastroenterol 38: 392–398, 2003.[CrossRef][ISI][Medline]

123. Järhult J, Angerås T, Farnebo LO, Graffner H, and Hamberger B. Elevated plasma levels of noradrenaline in duodenal ulcer. World J Surg 7: 385–389, 1983.[ISI][Medline]

124. Jaup EA, Timar Peregrin A, Jodal M, and Lundgren O. Nervous control of alkaline secretion in the duodenum as studied by the use of cholera toxin in the anaesthetized rat. Acta Physiol Scand 162: 165–174, 1998.[CrossRef][ISI][Medline]

125. Johansson B, Casselbrant A, Holm M, von Bothmer C, Johansson BR, and Fändriks L. Effects of hypovolaemia on acid-induced duodenal mucosal damage in the rat. Acta Physiol Scand 171: 43–50, 2001.[CrossRef][ISI][Medline]

126. Johansson B, Holm M, Ewert S, Casselbrant A, Pettersson A, and Fändriks L. Angiotensin II type 2 receptor-mediated duodenal mucosal alkaline secretion in the rat. Am J Physiol Gastrointest Liver Physiol 280: G1254–G1260, 2001.[Abstract/Free Full Text]

127. Johansson M, Synnerstad I, and Holm L. Acid transport through channels in the mucous layer of rat stomach. Gastroenterology 119: 1297–1304, 2000.[ISI][Medline]

128. Jönson C, Tunbäck-Hanson P, and Fändriks L. Splanchnic nerve activation inhibits the increase in duodenal HCO3 secretion induced by luminal acidification in the rat. Gastroenterology 96: 45–49, 1989.[ISI][Medline]

129. Joo NS, London RM, Kim HD, Forte LR, and Clarke LL. Regulation of intestinal Cl and HCO3 secretion by uroguanylin. Am J Physiol Gastrointest Liver Physiol 274: G633–G644, 1998.[Abstract/Free Full Text]

130. Jordan N, Newton J, Pearson J, and Allen A. A novel method for the visualization of the in situ mucus layer in rat and man. Clin Sci (Lond) 95: 97–106, 1998.[ISI][Medline]

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

132. Kim Y, Gum J, and Brockhausen I. Mucin glycoproteins in neoplasia. Glyconj J 13: 693–707, 1996.

133. Kindon H, Pothoulakis C, Thim L, Lynch-Devaney K, and Podolsky DK. Trefoil protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology 109: 516–523, 1995.[ISI][Medline]

134. Kirchgessner AL. Orexins in the brain-gut axis. Endocr Rev 23: 1–15, 2002.[Abstract/Free Full Text]

135. Kivilaakso E. High plasma HCO3 protects gastric mucosa against acute ulceration in the rat. Gastroenterology 81: 921–927, 1981.[ISI][Medline]

136. Kivilaakso E and Flemström G. Surface pH gradient in gastroduodenal mucosa. Scand J Gastroenterol 19, Suppl 105: 50–52, 1984.

137. Kiviluoto T, Ahonen M, Bäck N, Häppölä O, Mustonen H, Paimela H, and Kivilaakso E. Pre-epithelial mucus-bicarbonate layer protects against intracellular acidosis in acid-exposed gastric mucosa. Am J Physiol Gastrointest Liver Physiol 264: G57–G63, 1993.[Abstract/Free Full Text]

138. Kiviluoto T, Paimela H, Mustonen H, and Kivilaakso E. Intracellular pH in isolated Necturus antral mucosa exposed to luminal acid. Gastroenterology 98: 901–908, 1990.[ISI][Medline]

139. Knutson L and Flemström G. Duodenal mucosal bicarbonate secretion in man. Stimulation by acid and inhibition by the {alpha}2-adrenoceptor agonist clonidine. Gut 30: 1708–1715, 1989.[Abstract]

140. Knutson TW, Koss MA, Hogan DL, Isenberg JI, and Knutson L. Acetazolamide inhibits basal and stimulated HCO3 secretion in the human proximal duodenum. Gastroenterology 108: 102–107, 1995.[ISI][Medline]

141. Konturek SJ, Bilski J, Tasler J, and Laskiewicz J. Gastroduodenal alkaline response to acid and taurocholate in conscious dogs. Am J Physiol Gastrointest Liver Physiol 247: G149–G154, 1984.[Abstract/Free Full Text]

142. Konturek SJ, Bilski J, Tasler J, and Laskiewicz J. Gut hormones in stimulation of gastroduodenal alkaline secretion in conscious dogs. Am J Physiol Gastrointest Liver Physiol 248: G687–G691, 1985.[Abstract/Free Full Text]

143. Konturek SJ, Tasler J, Bilski J, and Kania J. Prostaglandins and alkaline secretion from oxynthic, antral and duodenal mucosa in the dog. Am J Physiol Gastrointest Liver Physiol 245: G539–G546, 1983.[Abstract/Free Full Text]

144. Krause WJ. Brunner's glands: a structural, histochemical and pathological profile. Prog Histochem Cytochem 35: 259–367, 2000.[Medline]

145. Kukkonen JP, Holmqvist T, Ammoun S, and Åkerman KE. Functions of the orexinergic/hypocretinergic system. Am J Physiol Cell Physiol 283: C1567–C1591, 2002.[Abstract/Free Full Text]

146. Lacy E and Ito S. Rapid epithelial restitution of the rat gastric mucosa after ethanol injury. Lab Invest 51: 573–585, 1984.[ISI][Medline]

147. Larsen KR, Moore JG, and Dayton MT. Circadian rhythms of acid and bicarbonate efflux in fasting rat stomach. Am J Physiol Gastrointest Liver Physiol 260: G610–G614, 1991.[Abstract/Free Full Text]

148. Larsen KR, Moore JG, and Dayton MT. Circadian rhythms of gastric mucus efflux and residual mucus gel in the fasting rat stomach. Dig Dis Sci 36: 1550–1555, 1991.[ISI][Medline]

149. Larson GM, Jedstedt G, Nylander O, and Flemström G. Intracerebral adrenoceptor agonists influence rat duodenal mucosal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 271: G831–G840, 1996.[Abstract/Free Full Text]

150. Lefebvre O, Chenard MP, Masson R, Linares J, Dierich A, LeMeur M, Wendling C, Tomasetto C, Chambon P, and Rio MC. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 274: 259–262, 1996.[Abstract/Free Full Text]

151. Lenz HJ. Regulation of duodenal bicarbonate secretion during stress by corticotropin-releasing factor and {beta}-endorphin. Proc Natl Acad Sci USA 86: 1417–1420, 1989.[Abstract]

152. Lenz HJ, Vale WW, and Rivier JE. TRH-induced vagal stimulation of duodenal HCO3 mediated by VIP and muscarinic pathways. Am J Physiol Gastrointest Liver Physiol 257: G677–G682, 1989.[Abstract/Free Full Text]

153. Lichtenberger L. The hydrophobic barrier properties of gastrointestinal mucus. Annu Rev Physiol 57: 565–583, 1995.[CrossRef][ISI][Medline]

154. Lichtenberger L, Graziani LA, Dial EJ, Butler BD, and Hills BA. Role of surface active phospholipids in cytoprotection. Science 219: 1327–1329, 1983.[ISI][Medline]

155. Lidell M, Johansson MEV, Morgelin M, Asker N, Gum JR, Kim YS, and Hansson GC. The recombinant C terminus of the human MUC2 mucin forms dimers in Chinese hamster ovary cells and heterodimers with full length Muc 2 in LS 174T cells. Biochem J 372: 335–345, 2003.[CrossRef][ISI][Medline]

156. Lugea A, Antolin M, Mourelle M, Guraner F, and Malagelada J. Deranged hydrophobic barrier of gastroduodenal mucosa after parenteral nonsteroidal anti-inflammatory drugs. Gastroenterology 112: 1931–1939, 1997.[ISI][Medline]

157. Lugea A, Mourelle M, Domingo A, Salas A, Guraner F, and Malagelada JR. Epidermal growth factor increases surface hydrophobicity and resistance to acid in the rat duodenum. Am J Physiol Gastrointest Liver Physiol 280: G774–G779, 2001.[Abstract/Free Full Text]

158. Malinovskaya NK, Komarov FI, Rapoport SI, Voznesenskaya LA, and Wetterberg L. Melatonin production in patients with duodenal ulcer. Neuroendocrinol Lett 22: 109–117, 2001.[ISI][Medline]

159. 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]

160. Mellander A and Sjövall H. Indirect evidence for cholinergic inhibition of intestinal bicarbonate absorption in humans. Gut 44: 353–360, 1999.[Abstract/Free Full Text]

161. Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. Mouse down-regulated in adenoma (DRA) is an intestinal Cl/HCO3 exchanger and is up-regulated in colon of mice lacking the NHE3 Na+/H+ exchanger. J Biol Chem 274: 22855–22861, 1999.[Abstract/Free Full Text]

162. Mertz-Nielsen A, Hillingsø J, Bukhave K, and Rask-Madsen J. Indomethacin decreases gastroduodenal mucosal bicarbonate secretion in humans. Scand J Gastroenterol 30: 1160 1165, 1995.[ISI][Medline]

163. Mertz-Nielsen A, Hillingsø J, Frøkiær H, Bukhave K, and Rask-Madsen J. Gastric bicarbonate secretion and release of prostaglandin E2 are increased in duodenal ulcer patients but not in Helicobacter pylori-positive healthy subjects. Scand J Gastroenterol 31: 38–43, 1996.[ISI][Medline]

164. Moniaux N, Escande F, Porchet N, Aubert JP, and Batra SK. Structural organization and classification of the human mucin genes. Front Biosci 6: D1192–D1206, 2001.[ISI][Medline]

165. Moore BA, Kim D, and Vanner S. Neural pathways regulating Brunner's gland secretion in guinea pig duodenum in vitro. Am J Physiol Gastrointest Liver Physiol 279: G910–G917, 2000.[Abstract/Free Full Text]

166. Morris G, Harding RK, and Wallace JL. A functional-model for extracellular gastric mucus in the rat. Virchows Arch 46: 239–251, 1984.

167. Newton J, Allen A, Westley BM, and May FEB. The human trefoil peptide, TFF1, is present in different molecular forms that are intimately associated with the adherent mucus gel in normal stomach. Gut 46: 312–320, 2000.[Abstract/Free Full Text]

168. Newton J, Jordan N, Oliver L, Strugala V, Pearson JP, James OJ, and Allen A. Helicobacter pylori in vivo causes structural changes in the adherent gastric mucus layer but barrier function is not compromised. Gut 43: 470–475, 1998.[Abstract/Free Full Text]

169. Nicholas C, Desai M, Vadgama P, McDonnell MB, and Lucas S. pH dependence of hydrochloric acid diffusion through gastric mucus: correlation with diffusion through a water layer using a membrane-mounted glass pH electrode. Analyst 116: 463–467, 1991.[CrossRef][ISI][Medline]

170. 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]

171. Nishizaki Y, Guth PH, Quintero E, Bover J, Del Rivero M, and Kaunitz JD. Prostaglandin E2 enhances gastric defense-mechanisms against acid injury in uremic rats. Gastroenterology 107: 1382–1389, 1994.[ISI][Medline]

172. Nylander O, Hällgren A, and Sababi M. COX inhibition excites enteric nerves that affect motility, alkaline secretion, and permeability in rat duodenum. Am J Physiol Gastrointest Liver Physiol 281: G1169–G1178, 2001.[Abstract/Free Full Text]

173. Nylander O, Kvietys P, and Granger DN. Effects of hydrochloric acid on duodenal and jejunal mucosal permeability in the rat. Am J Physiol Gastrointest Liver Physiol 257: G653–G660, 1989.[Abstract/Free Full Text]

174. Nylander O, Pihl L, and Perry M. Hypotonicity-induced increases in duodenal mucosal permeability facilitates adjustment of luminal osmolality. Am J Physiol Gastrointest Liver Physiol 285: G360–G370, 2003.[Abstract/Free Full Text]

175. Nylander O, Wilander E, Larson GM, and Holm L. Vasoactive intestinal polypeptide reduces hydrochloric acid-induced duodenal mucosal permeability. Am J Physiol Gastrointest Liver Physiol 264: G272–G279, 1993.[Abstract/Free Full Text]

176. 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]

177. Ovesen L, Bendtsen F, Tage-Jensen U, Pedersen NT, Gram BR, and Rune SJ. Intraluminal pH in the stomach, duodenum, and proximal jejunum in normal subjects and patients with exocrine pancreatic insufficiency. Gastroenterology 90: 958–962, 1986.[ISI][Medline]

178. Paimela H, Kiviluoto T, Mustonen H, Sipponen P, and Kivilaakso E. Tolerance of rat duodenum to luminal acid. Dig Dis Sci 35: 1244–1248, 1990.[ISI][Medline]

179. Patronella C, Vanek I, and Bowen JC. In vivo measurement of gastric mucus pH in canines: effect of high luminal acidity and prostaglandin E2. Gastroenterology 95: 612–618, 1988.[ISI][Medline]

180. Pavlov J. Die Arbeit der Verdaungsdrüsen. Wiesbaden, Germany: Bergman, 1898.

181. Pearson J, Ward RA, Allen A, Roberts NG, and Taylor W. Mucus degradation by pepsin: comparison of mucolytic activity of human pepsin 1 and pepsin 3: implications for peptic ulceration. Gut 27: 243–248, 1986.[Abstract]

182. Pérez JF, Ruiz MC, and Michelangeli F. Simultaneous measurement and imaging of intracellular Ca2+ and H+ transport in isolated rabbit gastric glands. J Physiol 537: 735–745, 2001.[Abstract/Free Full Text]

183. Perez-Vilar J and Hill R. The structure and assembly of secreted mucins. J Biol Chem 274: 31751–31754, 1999.[Free Full Text]

184. Phillipson M, Atuma C, Henriksnäs J, and Holm L. The importance of mucus and bicarbonate transport in preservation of gastric juxtamucosal pH. Am J Physiol Gastrointest Liver Physiol 282: G211–G219, 2002.[Abstract/Free Full Text]

185. Poulsom R and Wright NA. Trefoil peptides: a newly recognized family of epithelial mucin associated molecules. Am J Physiol Gastrointest Liver Physiol 265: G205–G213, 1993.[Abstract/Free Full Text]

186. Pratha VS, Hogan DL, Mårtensson BA, Bernard J, Zhou R, and Isenberg JI. Identification of transport abnormalities in duodenal mucosa and duodenal enterocytes from patients with cystic fibrosis. Gastroenterology 118: 1051–1060, 2000.[ISI][Medline]

187. Proetorius J, Andreasen D, Jensen BL, Ainsworth MA, Friis UG, and Johansen T. NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells. Am J Physiol Gastrointest Liver Physiol 278: G197–G206, 2000.[Abstract/Free Full Text]

188. Proetorius J, Friis UG, Ainsworth MA, Schaffalitzky de Muckadell OB, and Johansen T. The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells. Acta Physiol Scand 174: 327–236, 2002.[CrossRef][ISI][Medline]

189. Proetorius J, Hager H, Nielsen S, Aalkjoer C, Friis UG, Ainsworth MA, and Johansen T. Molecular and functional evidence for electrogenic and electroneutral Na+-HCO3 cotransporters in murine duodenum. Am J Physiol Gastrointest Liver Physiol 280: G332–G343, 2001.[Abstract/Free Full Text]

190. Quigley EMM and Turnberg LA. pH of the microclimate lining human gastric and duodenal mucosa in vivo. Studies in control subjects and in duodenal ulcer patients. Gastroenterology 92: 1876–1884, 1987.[ISI][Medline]

191. Ranta-Knuuttila T, Mustonen H, and Kivilaakso E. Topical prostaglandin E2 protects isolated gastric mucosa against acidified taurocholate-, but not ethanol- or aspirin-induced injury. Dig Dis Sci 45: 99–104, 2000.[CrossRef][ISI][Medline]

192. Rao SP, Sellers Z, Crombie DL, Hogan DL, Mann EA, Childs D, Keely S, Sheil-Puopolo M, Giannella RA, Barrett KE, Isenberg JI, and Pratha VS. A role for guanylate cyclase C in acid stimulated duodenal mucosal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 286: G95–G101, 2004.[Abstract/Free Full Text]

193. Reichstein BJ and Cohen MM. Effect of acetazolamide on rat gastric mucosal protection and stimulated bicarbonate secretion with 16,16-dimethyl prostaglandin E2. J Lab Clin Med 104: 797–804, 1984.[ISI][Medline]

194. Reimer R, Odes HS, Muallem R, Schwenk M, Beil W, and Sewing KF. Cyclic adenosine monophosphate is the second messenger of prostaglandin E2- and vasoactive intestinal polypeptide-stimulated active bicarbonate secretion by guinea-pig duodenum. Scand J Gastroenterol 29: 153–159, 1994.[ISI][Medline]

195. Ross I, Bahari HMM, and Turnberg LA. The pH gradient across mucus adherent to rat fundic mucosa in vivo and effect of potential damaging agents. Gastroenterology 81: 713–718, 1981.[ISI][Medline]

196. Rossmann H, Bachmann O, Vieillard-Baron D, Gregor M, and Seidler U. Na+/HCO3 cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells. Gastroenterology 116: 1389–1398, 1999.[ISI][Medline]

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

198. Säfsten B and Flemström G. Dopamine and vasoactive intestinal peptide stimulate cyclic adenosine-3',5'-monophosphate formation in isolated rat villus and crypt duodenocytes. Acta Physiol Scand 149: 67–75, 1993.[ISI][Medline]

199. Säfsten B, Jedstedt G, and Flemström G. Effects of diazepam and Ro 15-1788 on duodenal bicarbonate secretion in the rat. Gastroenterology 101: 1031–1038, 1991.[ISI][Medline]

200. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, and Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92: 573–585, 1998.[ISI][Medline]

201. Samloff MI. Peptic ulcer: the many proteinases of aggression. Gastroenterology 96: 586–595, 1989.[ISI][Medline]

202. Sands B and Podolsky DK. The trefoil peptide family. Annu Rev Physiol 58: 253–273, 1996.[CrossRef][ISI][Medline]

203. Sawaguchi A, Ishihara K, Kawano J, Oinuma T, Hotta K, and Suganuma T. Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric gland processed by high-pressure freezing/freeze substitution. J Histochem Cytochem 50: 223–234, 2002.[Abstract/Free Full Text]

204. Schade C, Flemström G, and Holm L. Hydrogen ion concentration in the mucus layer on top of acid- stimulated and acid-inhibited rat gastric mucosa. Gastroenterology 107: 180–188, 1994.[ISI][Medline]

205. Schierbeck N. Ueber Kohlensäure im Ventrikel. Skand Arch Physiol 3: 437–474, 1892.

206. Schreiber S, Nguyen TH, Stuben M, and Scheid P. Demonstration of a pH gradient in the gastric gland of the acid-secreting guinea pig mucosa. Am J Physiol Gastrointest Liver Physiol 279: G597–G604, 2000.[Abstract/Free Full Text]

207. Schreiber S and Scheid P. Gastric mucus of the guinea pig: proton carrier and diffusion barrier. Am J Physiol Gastrointest Liver Physiol 272: G63–G70, 1997.[Abstract/Free Full Text]

208. Schwarz K. Ueber penetrierende Magen und Jejunalgeschwüre. Beitr Klin Chir 67: 96–128, 1910.

209. Seidler U, Blumenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans M, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3 secretion. J Physiol 505: 411–423, 1997.[Abstract]

210. Sellers LA, Allen A, and Bennett MK. Formation of fibrin based gelatinous coat over repairing rat gastric epithelium after cute ethanol damage: interaction with adherent mucus. Gut 28: 835–843, 1987.[Abstract]

211. Sellers LA, Allen A, Morris E, and Ross-Murphy SB. Mucus glycoprotein gels. Role of glycoprotein polymeric structure and carbohydrate side-chains in gel-formation. Carbohydr Res 178: 93–110, 1988.[CrossRef][ISI][Medline]

212. Shorrock CJ, Prescott JR, and Rees WDW. The effects of indomethacin on gastroduodenal morphology and mucosal pH gradient in the healthy human stomach. Gastroenterology 199: 334–339, 1990.

213. Silen W and Ito S. Mechanisms for rapid re-epithelialisation of the gastric mucosal surface. Annu Rev Physiol 47: 217–229, 1985.[CrossRef][ISI][Medline]

214. Simpson J, Merhav A, and Silen W. Alkaline secretion by amphibian duodenum I. General characteristics. Am J Physiol Gastrointest Liver Physiol 240: G401–G408, 1981.[Abstract/Free Full Text]

215. Sjöblom M and Flemström G. Melatonin in duodenal lumen is a potent stimulant of mucosal bicarbonate secretion. J Pineal Res 34: 288–293, 2003.[CrossRef][ISI][Medline]

216. Sjöblom M and Flemström G. Central nervous {alpha}1-adrenoceptor stimulation induces duodenal luminal release of melatonin. J Pineal Res 36: 1–6, 2004.[CrossRef][ISI][Medline]

217. Sjöblom M, Jedstedt G, and Flemström G. Peripheral melatonin mediates neural stimulation of duodenal mucosal bicarbonate secretion. J Clin Invest 108: 625–633, 2001.[Abstract/Free Full Text]

218. Sjöblom M, Säfsten B, and Flemström G. Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes. Am J Physiol Gastrointest Liver Physiol 284: G1034–G1044, 2003.[Abstract/Free Full Text]

219. Sjövall H, Forssell H, Häggendal J, and Olbe L. Reflex sympathetic activation in humans is accompanied by inhibition of gastric HCO3 secretion. Am J Physiol Gastrointest Liver Physiol 255: G752–G758, 1988.[Abstract/Free Full Text]

220. Snary D, Allen A, and Pain RH. Structural studies on gastric mucoproteins lowering of molecular weight after reduction with 2-mercaptoethanol. Biochem Biophys Res Commun 40: 844–851, 1970.[ISI][Medline]

221. Solcia E, Vasallo G, and Sampietro R. Endocrine cells in the antro-pyloric mucosa of the stomach. Z Zellforsch 81: 474–486, 1967.[CrossRef][ISI][Medline]

222. Spiegel S, Phillipper M, Rossmann H, Riederer B, Gregor M, and Seidler U. Independence of apical Cl/HCO3 exchange and anion conductance in duodenal HCO3 secretion. Am J Physiol Gastrointest Liver Physiol 285: G887–G897, 2003.[Abstract/Free Full Text]

223. Spychal R, Goggin PM, Marrero JM, Saverymuttu SH, Yu CW, Corbishley CM, Maxwell JD, and Norfield TC. Surface hydrophobicity of the gastric mucosa in peptic ulcer disease. Relationship to gastritis and Campylobacter pylori infection. Gastroenterology 98: 1250–11254, 1990.[ISI][Medline]

224. Spychal R, Marrero JM, Saverymuttu SH, and Norfield TC. Measurement of hydrophobicity of human gastrointestinal mucosa. Gastroenterology 97: 104–111, 1989.[ISI][Medline]

225. Synnerstad I, Ekblad E, Sundler F, and Holm L. Gastric mucosal smooth muscles may explain oscillations in glandular pressure: role of vasoactive intestinal peptide. Gastroenterology 114: 284–294, 1998.[ISI][Medline]

226. Synnerstad I and Holm L. Prostaglandin E2 and aggressive factors increase the gland luminal pressure in the rat gastric mucosa in vivo. Gastroenterology 114: 1276–1286, 1998.[ISI][Medline]

227. Synnerstad I, Johansson M, Nylander O, and Holm L. Intraluminal acid and gastric mucosal integrity: the importance of blood-borne bicarbonate. Am J Physiol Gastrointest Liver Physiol 280: G121–G129, 2001.[Abstract/Free Full Text]

228. Synnerstad I, Persson AEG, and Holm L. Effect of inhibition of pentagastrin-stimulated acid secretion on gastric mucosal gland luminal pressure. Acta Physiol Scand 160: 103–111, 1997.[CrossRef][ISI][Medline]

229. Takeuchi K, Furukawa O, Tanaka H, and Okabe S. A new model of duodenal ulcers induced in rats by indomethacin plus histamine. Gastroenterology 90: 636–645, 1986.[ISI][Medline]

230. Takeuchi K, Magee D, Critchlow J, Matthews J, and Silen W. Studies of the pH gradient and thickness of frog gastric mucus gel. Gastroenterology 84: 331–340, 1983.[ISI][Medline]

231. Takeuchi K, Merhav A, and Silen W. Mechanism of luminal alkalinization by bullfrog fundic mucosa. Am J Physiol Gastrointest Liver Physiol 243: G377–G388, 1982.[Abstract/Free Full Text]

232. Takeuchi K, Takehara K, Kato S, and Yagi K. PACAPs stimulate duodenal bicarbonate secretion at PACAP receptors in the rat. Am J Physiol Gastrointest Liver Physiol 272: G646–G653, 1997.[Abstract/Free Full Text]

233. 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]

234. Takeuchi K, Ukawa H, Kato S, Furukawa O, Arachi H, Sugimoto Y, Ichikawa A, Ushikobo E, and Narumiya S. Impaired duodenal bicarbonate secretion and mucosal integrity in mice lacking prostaglandin E-receptor subtype EP3. Gastroenterology 117: 1128–1135, 1999.[ISI][Medline]

235. Takeuchi K, Yagi K, Kato S, and Ukawa H. Roles of prostaglandin E-receptor subtypes in gastric and duodenal bicarbonate secretion in rats. Gastroenterology 113: 1553–1559, 1997.[ISI][Medline]

236. Tanaka S, Meiselman HHJ, Engel E, Guth PH, Furukawa O, Wenby RB, Lee J, and Kaunitz JD. Regional differences of H+, HCO3, and CO2 diffusion through native porcine gastroduodenal mucus. Dig Dis Sci 47: 967–973, 2002.[CrossRef][ISI][Medline]

237. Tanaka S, Podolsky D, Engel E, Guth PH, and Kaunitz JD. Human spasmolytic polypeptide decreases proton permeation through gastric mucus. Am J Physiol Gastrointest Liver Physiol 272: G1473–G1480, 1997.[Abstract/Free Full Text]

238. Tanaka S, Taché Y, Keneko H, Guth PH, and Kaunitz JD. Central vagal activation increases mucus gel thickness and surface intracellular pH in the rat stomach. Gastroenterology 112: 409–417, 1997.[ISI][Medline]

239. Tani S, Suzuki T, Kano S, Tanaka T, Sunaga K, Morishige R, and Tsuda T. Mechanisms of gastric mucus secretion from cultured rat gastric epithelial cells induced by carbachol, cholecystokinin octapeptide, secretin, and prostaglandin E2. Biol Pharm Bull 25: 14–18, 2002.[CrossRef][ISI][Medline]

240. Taupin D and Podolsky DK. Trefoil factors initiators of mucosal healing. Nat Rev Mol Cell Biol 4: 721, 2003.[CrossRef][ISI][Medline]

241. Taylor C, Allen A, Dettmar PW, and Pearson JP. The gel matrix of gastric mucus is maintained by a complex interplay of transient and nontransient associations. Biomacromolecules 4: 922–927, 2003.[CrossRef][ISI][Medline]

242. Taylor C, Allen A, Dettmar PW, and Pearson JP. Two rheologically different gastric mucus secretions with different putative functions. Biochim Biophys Acta 1674: 131–138, 2004.[ISI][Medline]

243. Taylor W. Pepsins of patients with peptic ulcer. Nature 227: 76–77, 1970.[ISI][Medline]

244. Teorell T. A method of studying conditions within diffusion layers. J Biol Chem 113: 735–748, 1936.

245. Thangarajah H, Wong A, Chow DC, Crothers JM, and Forte JG. Gastric H-K-ATPase and acid-resistant surface proteins. Am J Physiol Gastrointest Liver Physiol 282: G953–G961, 2002.[Abstract/Free Full Text]

246. Thim L, Madsen F, and Poulsen SS. Effect of trefoil factors on the viscoelastic properties of mucus gels. Eur J Clin Invest 32: 519–527, 2002.[CrossRef][ISI][Medline]

247. Tobey N, Hosseini SS, Caymaz-Bor C, Wyatt HR, Orlando GS, and Orlando RC. The role of pepsin in acid injury to esophageal epithelium. Am J Gastroenterol 96: 3062–3070, 2001.[ISI][Medline]

248. Tomasetto C, Masson R, Linares JL, Wendling C, Lefebvre O, Chenard MP, and Rio MC. pS2/TFF1 interacts directly with the VWFC cysteine-rich domains of mucins. Gastroenterology 118: 70–80, 2000.[ISI][Medline]

249. Tuo BG and Isenberg JI. Effect of 5-hydroxytryptamine on duodenal mucosal bicarbonate secretion in mice. Gastroenterology 125: 805–814, 2003.[CrossRef][ISI][Medline]

250. Tuo BG, Sellers Z, Paulus P, Barrett KE, and Isenberg JI. 5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice. Am J Physiol Gastrointest Liver Physiol 286: G444–G451, 2004.[Abstract/Free Full Text]

251. Ulrich CD. Bicarbonate secretion and CFTR: continuing the paradigm shift. Gastroenterology 118: 1258–1261, 2000.[ISI][Medline]

252. Van Seuningen I, Pigny P, Perrais M, Porchet N, and Aubert JP. Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer? Front Biosci 6: D1216–D1234, 2001.[ISI][Medline]

253. Von Bothmer C, Edebo A, Lönnroth H, Olbe L, Pettersson A, and Fändriks L. Helicobacter pylori infection inhibits antral mucosal nitric oxide production in humans. Scand J Gastroenterol 37: 404–408, 2002.[CrossRef][ISI][Medline]

254. Walker NM, Flagella M, Gawenis LR, Shull GE, and Clarke LL. An alternate pathway of cAMP-stimulated Cl secretion across the NKCC1-null murine duodenum. Gastroenterology 123: 531–541, 2002.[CrossRef][ISI][Medline]

255. Wallace JL. Gastric resistance to acid: is the "mucus-bicarbonate barrier" functionally redundant? Am J Physiol Gastrointest Liver Physiol 256: G31–G38, 1989.[Abstract/Free Full Text]

256. Wallace JL and Ma L. Inflammatory mediators in gastrointestinal defense and injury. Exp Biol Med 2261003–1015, 2001.

257. Wang Z, Petrovic S, Mann E, and Soleimani M. Identification of an apical Cl/HCO3 exchanger in the small intestine. Am J Physiol Gastrointest Liver Physiol 282: G573–G579, 2002.[Abstract/Free Full Text]

258. Wenzl E, Feil W, Starlinger M, and Schiessel R. Alkaline secretion. A protective mechanism against acid injury in rabbit duodenum. Gastroenterology 92: 709–715, 1987.[ISI][Medline]

259. Williams S and Turnberg LA. Retardation of acid diffusion by pig gastric mucus: a potential role in mucosal protection. Gastroenterology 79: 299–304, 1980.[ISI][Medline]

260. Williams S and Turnberg LA. Demonstration of a pH gradient across mucus adherent to rabbit gastric mucosa: evidence for mucus bicarbonate barrier. Gut 22: 94–96, 1981.[Abstract]

261. Wolosin JD, Thomas FJ, Hogan DL, Koss MA, O'Dorisio TM, and Isenberg JI. The effect of vasoactive intestinal peptide, secretin, and glucagon on human duodenal bicarbonate secretion. Scand J Gastroenterol 24: 151–157, 1989.[Medline]

262. Xu J, Barone S, Petrovic S, Wang Z, Seidler U, Riederer B, Ramaswamy Pradeep K, Dudeja PK, Shull GE, and Soleimani M. Identification of an apical Cl/HCO3 exchanger in gastric surface mucous and duodenal villus cells. Am J Physiol Gastrointest Liver Physiol 285: G1225–G1234, 2003.[Abstract/Free Full Text]

263. Yao B, Hogan DL, Bukhave K, Koss MA, and Isenberg JI. Bicarbonate transport by rabbit duodenum in vitro: effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate. Gastroenterology 105: 957–959, 1993.[ISI][Medline]

264. Younan F, Pearson JP, Allen A, and Venables CW. Changes in the structure of the mucus gel on the mucosal surface of the stomach in association with peptic ulcer disease. Gastroenterology 82: 827–831, 1982.[ISI][Medline]