Departments of 1 Physiology and 3 Pathology, Uppsala University, SE-751 23 Uppsala, Sweden; 2 Swedish Institute for Infectious Disease Control, SE-105 21 Stockholm, Sweden; 4 Department of Physiological Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom
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ABSTRACT |
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The aim of this
study was to investigate the permeability of the adherent mucus gel
layer in rat duodenum in vivo to macromolecules applied in the lumen.
Rats were anesthetized with thiobarbiturate, and the duodenum was
perfused with isotonic NaCl solution containing large-molecular-size
secretagogues. Effects on mucosal HCO3 secretion and blood-to-lumen
51chromium-labeled EDTA clearance
were used as indexes that compounds had migrated across the mucus
layer. Exposure to a low concentration of papain (10 U/100 ml) for 30 min removed the mucus layer without damage to the epithelium and
induced or markedly enhanced HCO
3 secretory responses to cholera toxin (molecular mass of 85 kDa) or glucagon (3.5 kDa). Water extracts from a VacA
cytotoxin (89 kDa) producing Helicobacter
pylori strain, but not from a toxin-negative isogenic
mutant, caused a small increase in
HCO
3 secretion but only after the
mucus layer had been removed by papain. The duodenal surface mucus gel
thus significantly restricts migration of macromolecules to the
duodenal surface. Release of bacterial toxins at the cell-mucus
interface may enhance or be a prerequisite for their effects on the
gastrointestinal mucosa.
cholera toxin; chromium-labeled EDTA clearance; glucagon; duodenal bicarbonate secretion; Helicobacter pylori; prostaglandin E2; VacA cytotoxin
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INTRODUCTION |
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THE GASTRODUODENAL MUCOSA is covered by an adherent
layer of visco-elastic mucus gel, which provides a physical barrier
between the apical cell surfaces and the lumen. The viscous and
gel-forming properties of this mucus gel are derived from mucin
glycoproteins, which constitute ~3-5% of the gel by weight,
with the remaining 95% being water together with small amounts of
lipids, nucleic acids, and other proteins, including immunoglobulins
(1). In stomach, evidence from several sources, both in vivo and in
vitro, shows that mucus forms a continuous, stable, and firm gel layer adherent to the surface epithelium (20, 21, 32, 33).
HCO3 secretion maintains the pH in the
mucus gel adjacent to the gastric (10, 27, 32) and duodenal (13, 27,
29) epithelial surface at a pH value considerably higher than that in
the gastric lumen. Furthermore, the adherent mucus layer in the stomach
prevents access of the macromolecule pepsin from the lumen to the
gastric epithelial surface (4). Transport of acid and pepsin from the crypts into the gastric lumen, in contrast, is not prevented (15) and
appears to occur through channels (32) in the mucus gel formed by the
secretory hydrostatic pressure (36).
The thickness of the mucus gel layer in the duodenum is, despite the marked differences in epithelial surface topology, similar to that in the stomach. The mean thickness measured with microelectrodes in rat duodenum in vivo amounted to 280 µm (29) and that measured on washed mucosa in vitro amounted to ~90 µm (3). By preventing or restricting migration of macromolecules, the mucus layer may provide important mechanisms for protection against toxins ingested with food, luminal proteinases, and other putative mucosa-damaging agents. In the case of toxins produced by bacteria living in the mucus layer, their release at the mucus-cell interface could enhance their deleterious effects on the mucosa. Furthermore, the mucus layer could have implications for oral vaccines by restricting the migration of these macromolecular antigens from the intestinal lumen to the immunoactive cells at the epithelial surface and thus interfering with the induction of immunity.
Studies in vitro have suggested that the rate of diffusion of solutes through mucus gel progressively decreases with increasing molecular size (1, 7). The access of disaccharide and small peptide substrates to brush-border enzymes in intestinal loops in vitro has been shown to be reduced by the adherent mucus layer (34). Large-molecular-size proteins of several thousand molecular weight do not significantly diffuse through artificial mucus gel layers over several hours (2). However, the permeability of macromolecules through the intestinal mucus layer in vivo in intact animals has not been studied before.
The aim of the present investigation was therefore to develop a model
for studies of the migration of macromolecules across the duodenal
mucus gel in vivo. The concept of our approach was to use some
large-molecular-size toxins and secretagogues that should affect
duodenal mucosal HCO3 secretion and/or
mucosal permeability. Changes in the rate of
HCO
3 secretion and/or
51Cr-labeled EDTA permeability
were used as indexes of the migration across the mucus gel layer to the
epithelial surface of macromolecules instilled into the intestinal
lumen in vivo. The blood-to-lumen clearance of
51Cr-EDTA used here to estimate
permeability is, furthermore, a good estimate of mucosal integrity
(25). A method for removal of the adherent mucus gel in the duodenum by
mild treatment with papain was established. Macromolecular
toxins/secretagogues, and for comparison
PGE2, were applied luminally, and
experiments were performed in duodenum with an intact mucus gel and
after substantial removal of the mucus gel by papain. A preliminary
account of some of the results has been published in abstract form
(12).
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MATERIALS AND METHODS |
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Male F1 hybrids of Lewis × Dark Agouti rats (Animal Department, Biomedical Center, Uppsala, Sweden), weighing 200-300 g, were kept under standardized conditions of temperature (21-22°C) and light (12:12-h light-dark cycle) and given pelleted food (Ewos, Södertälje, Sweden) ad libitum. Before experiments, the animals were fasted overnight in groups of two or more in cages with mesh bottoms but had free access to drinking water. The operative procedures have been described before in detail (14, 25), and a summary and some modifications are provided here. To avoid stress, rats were anesthetized in the Animal Department by their regular keeper with an intraperitoneal injection of 120 mg/kg body wt sodium-5-ethyl-5-(1'-methylpropyl)-2-thiobarbituric acid (Inactin). Rats were tracheotomized with a tracheal cannula to facilitate respiration. An external jugular vein and femoral artery and vein were catheterized with PE-50 polyethylene catheters (Becton-Dickinson, Parsippany, NJ). The veins were used for infusion of 51Cr-EDTA and drug (hexamethonium) injections. For the continuous recording of systemic arterial blood pressure, the arterial catheter, containing heparin (20 IU/ml dissolved in saline), was connected to a pressure transducer (Gould Statham P23 ID, Oxnard, CA) operating a polygraph (polygraph 7D, Grass, Quincy, MA).
Subsequently, a laparotomy was performed, and the common bile duct was catheterized with a PE-10 polyethylene tube close to its entrance into the duodenum (2-3 mm) to avoid contamination with pancreatico-biliary secretions. A soft silicone tube (Silastic, 1 mm ID; Dow Corning, Midland, MI) was introduced into the mouth and pushed gently along the esophagus into the stomach and through the pylorus and secured by two ligatures 2-5 mm distal to the pylorus. A PE-320 cannula was inserted into the duodenum ~2.5-3.5 cm distal to the pylorus and secured by ligatures. The proximal duodenal cannula was connected to a peristaltic pump (Gilson Minipulse 3, Villiers, Le Bel, France), and continuous perfusion (0.4 ml/min) of the segment with isotonic NaCl solution was started. The effluent was collected in 10-min samples. Surgery was completed by closing the abdominal cavity with sutures, and the wound was covered with plastic foil. After surgery, the animals were allowed to recover for at least 45 min to stabilize cardiovascular, respiratory, and gastrointestinal functions. All experiments had been approved by the Uppsala University Ethical Committee for Experiments with Animals.
Luminal perfusions.
The macromolecules added to the duodenal luminal perfusate were chosen
on the basis that they were either established as or possible stimuli
or inhibitors of duodenal mucosal HCO3 secretion. Parenteral administration of the peptide
hormone glucagon (molecular mass of 3.5 kDa) increases the
HCO
3 secretion in all species tested
except humans (10, 11, 38). Luminally administered cholera toxin
[subunit A (molecular mass of 85 kDa)] is a
well-known stimulant of electrolyte secretion in more distal small
intestine (19). Infection with H. pylori has been proposed to inhibit duodenal mucosal
HCO
3 secretion in patients with
duodenal ulcer disease (16). The effects of water extracts of the VacA
cytotoxin (molecular mass of 89 kDa) producing reference strain CCUG
17874 and one cytotoxin-negative (VacA
) isogenic mutant were
tested in the present study. Furthermore, PGE2 (molecular mass of 335 Da), a
smaller and lipophilic molecule and a stimulant of duodenal
HCO
3 secretion in all species tested
in vivo and in vitro (10), was perfused luminally in some experiments.
Removal of the continuous mucus gel. Perfusion with papain-containing solution (10 U/100 ml papain, 5 mM L-cysteine, and NaCl to isotonicity; pH 7.3) for 30 min was used to remove the continuous surface mucus gel layer. To avoid effects on epithelial tight junctions, EDTA was not included in the perfusate. The papain was then removed (by 10 min of perfusion with isotonic NaCl alone), and perfusion was continued for another 40-120 min with NaCl solution containing purified cholera toxin, water extracts of H. pylori strains, the hormone glucagon, or PGE2.
The absence of a continuous mucus gel layer at the duodenal surface, as well as the absence of any gross mucosal damage, was confirmed at the end of all experiments involving exposure to papain. The duodenal segment under study and the just distal (not perfused) duodenal segment were both removed, and two unfixed specimens of each of these segments were used to measure adherent surface mucus gel thickness by light microscopy (21). The presence of a continuous mucus gel layer was confirmed in all duodena perfused with isotonic NaCl solution alone. For further and detailed morphological examination, the perfused segments from two to three duodena from all experimental groups were fixed in 10% neutral buffered formalin. Two longitudinal specimens were taken, embedded in paraffin, dehydrated, stained with hematoxylin-eosin, and examined for mucosal damage by an experienced pathologist who was unaware of the experimental protocol.Measurement of mucosal HCO3
secretion.
The rate of secretion was determined by back titration of 1-ml samples
of the solution to be infused and the effluent to pH 5.0 with 50 mM HCl
during continuous gassing with 100%
N2 (to remove
CO2), using pH stat equipment
(Radiometer, Copenhagen, Denmark). The amount of titratable base in
infused solutions containing protein was always <20% of the amount
of HCO
3 secreted by the duodenal
mucosa and was deducted from the measured rates. The pH electrode was
calibrated with standard buffers before the start of the titrations.
The rate of secretion was expressed as microequivalents of
HCO
3 secretion per centimeter of
intestine per hour
(µeq · cm
1 · h
1).
Measurement of mucosal permeability.
After completion of surgery,
51Cr-EDTA was administered
intravenously as a bolus of 75 µCi followed by a continuous infusion at a rate of 50 µCi/h. The radioactive isotope was diluted in a
Ringer-HCO3 solution and infused at a
rate of 1 ml/h (Harvard Apparatus, South Natick, MA). Forty-five
minutes was permitted for tissue equilibration of the
51Cr-EDTA. Three blood samples
(0.2 ml) were collected during the experiment, and the blood volume
loss was compensated for by injection of a 10% Ficoll solution. After
centrifugation, 50 µl of the plasma were removed for measurements of
radioactivity. The luminal perfusate and the plasma were analyzed for
51Cr activity in a gamma counter
(1282 Compugamma CS, Pharmacia, Uppsala, Sweden). A linear regression
analysis of the plasma samples was made to obtain a corresponding
plasma value for each effluent sample. The clearance of
51Cr-EDTA from blood to lumen was
calculated as described before (16) and was expressed as milliliters
per minute per 100 g wet tissue weight
(ml · min
1 · 100 g
1 wet tissue wt).
Macromolecules, drugs, and solutions.
Highly purified cholera toxin (no. 101B) was obtained from List
Biological Laboratories (Campbell, CA). Glucagon (porcine), PGE2, hexamethonium chloride,
crystallized papain (20-25 U/mg), and
L-cysteine (free base) were from
Sigma (St. Louis, MO). The anesthetic Inactin was from Research
Biochemicals International (Natick, MA), and
51Cr-EDTA was from DuPont NEN
(Boston, MA). All agents, except
PGE2, were dissolved in isotonic
saline at pH 7.4 on the day of use. PGE2 was dissolved in ethanol (5 mmol/l), and this stock solution was stored at 20°C. The
prostaglandin was added as a small amount (
15 µl/ml saline) from
the stock solution.
Statistical analyses.
All values are expressed as means ± SE, with
n representing the number of
experiments. Rates of duodenal HCO3 secretion in control animals perfused with isotonic NaCl alone remained
stable over the time periods used in the present experiments (up to 240 min). This was confirmed in animals with an intact surface mucus gel as
well as after removal of the mucus gel by papain. Statistical analyses
of the effects of luminal secretagogues were performed by ANOVA
followed by a Fisher's protected least-significant difference test,
comparing rates of HCO
3 secretion before and after addition of a potential secretagogue (repeated measures). Only one concentration of a secretagogue was tested in the
same animal, and two-way ANOVA was used (always stated in the text) to
compare effects in groups of animals exposed to different perfusate
concentrations of cholera toxin, glucagon, and
PGE2. A
P value of <0.05 was considered
significant. The mean arterial blood pressure was continuously
recorded, and HCO
3 secretion and
blood-to-lumen clearance of
51Cr-EDTA were measured at 10-min intervals.
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RESULTS |
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Removal of the surface mucus gel. The concentration of papain in the luminal perfusate (10 U/100 ml) and time of exposure (30 min) were kept low to avoid damage of the mucosa. Unfixed specimens of papain-exposed duodena were examined at the end of each experiment by light microscopy, enabling detection of the mucus gel layer (21). The continuity of the duodenal surface mucus gel was always broken after papain, with most of the surface being free of visible mucus. However, patches (20-50 µm length) of a thin (<20 µm) layer of adherent mucus could be observed after some of the experiments. No areas free of adherent mucus gel were observed in adjacent (just distal) nonperfused segments of duodenum from the same animals.
In contrast to papain-perfused duodenal segments, a continuous mucus layer, of minimum thickness >20 µm, was always observed covering perfused but not papain-exposed mucosa. Light microscopy of unfixed specimens does not allow detailed examination of mucosal cells. However, no damage to the villi or other pathological changes could be observed on examination of fixed specimens of duodenum from all papain-exposed groups (observed blind to the experimental protocol). Furthermore, after treatment with papain, mucosal HCO
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Effects of cholera toxin.
Cholera toxin is a well-known stimulus of NaCl and water secretion in
more distal small intestine (19, 22). Effects on the
HCO3 secretion by the duodenal mucosa
are shown in Fig. 1. In duodenum with an intact mucus gel, there was a
significant rise in secretion after exposure to the higher (8 µg/ml)
concentration of cholera toxin (P < 0.05 at 150 min, compared with basal rates in the same animals).
However, a marked (greater than twofold) response was observed only in
three of nine duodena. Exposure to a lower (2 µg/ml) concentration of
cholera toxin did not significantly increase (0.10 > P > 0.05) the
HCO
3 secretion.
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Effects of Helicobacter pylori extracts.
There is a clear association between the presence of peptic ulcer
disease and infection with H. pylori
in humans (5). Furthermore, basal HCO3
secretion and the ability of the duodenal mucosa to respond to luminal
acid with a rise in mucosal HCO
3
secretion are reduced in patients with duodenal ulcers (17, 24), and
secretion was recently reported to normalize, in part, after
eradication of the bacterium (16).
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Effects of glucagon.
Glucagon stimulates duodenal mucosal
HCO3 secretion when administered
parenterally in animals in vivo or to the serosal (blood) side of
duodenal mucosa in vitro (10, 11). Luminal administration of a high
concentration (15 µM) of this peptide induced a small but significant
(P < 0.05 at 90 min, compared with
basal rates in the same animals) increase in secretion (Fig.
5), which was reversible on removal of the
peptide from the luminal perfusate (not shown). A lower concentration of glucagon (0.15 µM) did not affect the
HCO
3 secretion. When papain had been
used to remove the continuous mucus gel (Fig. 5), the response to 15 µM glucagon was greatly enhanced (P < 0.05, comparison with intact mucus gel), but a significant response
to the lower glucagon concentration of 0.15 µM did not occur (0.10 > P >0.05, compared with basal
rates in the same animals). However, six of nine mucosae now showed a
small response to the 100-fold lower concentration of the peptide. No
changes in 51Cr-EDTA clearance
(not shown) occurred in these experiments.
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Effects of PGE2.
This lipophilic molecule is important in the physiological control of
the duodenal secretion, and exogenous E-type prostaglandins stimulate
secretion in all species tested (10). Luminal perfusion with 20 µM
PGE2 caused a rise in
HCO3 secretion (P < 0.05 at 60 min, compared with
basal rates in the same animal) in duodenum with an intact mucus gel
(Fig. 6) similar in size to that observed
before with this concentration of the prostaglandin (10, 11). Perfusion
with a higher concentration (75 µM) induced a greater response
(P < 0.05, compared with 20 µM).
Removal of the mucus gel by papain resulted in a more rapidly occurring
and greater (P < 0.05, compared with
intact mucus gel) response to the lower concentration of the
prostaglandin. No changes in mucosal permeability, measured as
51Cr-EDTA clearance, were
observed.
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DISCUSSION |
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The gastrointestinal mucosa provides a dynamic barrier between the host
and contents of the lumen, allowing the passage of certain molecules
into the body while restricting the entry of others. This function is
present throughout the consecutive regions of the gastrointestinal
tract, including the duodenum, but is faced with very different
challenges in the different regions. It protects the epithelium against
proteolytic enzymes and ingested bacteriotoxins and the body against
antigenic challenges (31). However, for ion-transporting enterocytes or
immunoactive cells (or receptors on neurons and/or paracrine cells
mediating responses) to respond to changes in composition of the
luminal bulk solution, they must first be able to recognize the
stimulant. The layer of adherent mucus gel is an important part of the
gastrointestinal mucosal barrier, but its permeability characteristics
have been only broadly defined. Studies measuring surface pH gradients
and epithelial HCO3 transport in
intact mucosa indicate that both
H+ and
HCO
3 rapidly diffuse through the
adherent mucus gel layer on top of gastric and duodenal mucosa in vivo (10, 13, 29). Studies of mucus gel in vitro have demonstrated that
H+ diffuse 4- to 14-fold
slower (26, 37) and HCO
3 diffuse
11-fold slower (23) in mucus than in an equivalent unstirred layer of
saline. These rates are still very fast, and the diffusion of
H+ and other monovalent ions
through the mucus gel layer is not thought to be significantly retarded
in physiological terms (2, 8). This is confirmed in the present studies
in which treatment with papain, shown to remove the mucus layer, did
not change the rate of HCO
3 secretion.
In contrast to smaller molecular solutes, studies of mucus gel in vitro
have suggested that the rate of diffusion of larger molecules across
the gel progressively decreases with increasing molecular size (1, 7).
In the present study, after removal of the duodenal mucus barrier by
papain, there was a significantly greater rise in
HCO3 secretion in response to luminal
perfusion with the probe macromolecules cholera toxin (molecular mass
of 85 kDa), glucagon (3.5 kDa), and H. pylori cytotoxin (89 kDa). The combined results
demonstrate that the gel layer at the duodenal surface provides a
significant barrier between the lumen and the underlying epithelium for
these large-molecular-weight HCO
3
secretagogues. The same concentrations of toxins or glucagon did not
affect mucosal blood-to-lumen
51Cr-EDTA clearance, indicating,
furthermore, that the increase in HCO
3
secretion reflects an increase in transcellular transport of
HCO
3 and not an increase in mucosal permeability.
In the case of cholera toxin and glucagon at higher concentrations,
some stimulation of HCO3 secretion
occurred without prior removal of the mucus gel layer and with the
mucus barrier visually intact on unfixed sections. Some macromolecules thus seem to penetrate across the mucus gel to the epithelial surface
provided that their luminal concentration is sufficiently high. This
would suggest that the overall cover of the mucus layer in the duodenum
is not totally impermeable to large-molecular-weight proteins and that
such molecules can gain limited access to the epithelium. The mucus gel
layer in rat duodenum in vivo can be locally almost completely removed
using suction with a small catheter coupled to a syringe (29). Shear
forces of the digestive processes, or perfusion of the duodenal lumen
as used in the present study, could cause thinning, leading to small
discontinuities in the mucus gel not apparent in unfixed sections of
washed mucosa. Small discontinuities in the gel layer in vivo could
also be lost by annealing of the mucus gel on sectioning of the mucosa.
However, there was clearly an increased stimulation of
HCO3 secretion after removal of the
surface mucus gel in response to a low concentration (20 µM) of
PGE2 (molecular mass of 335 Da).
This is interesting because it demonstrates that the duodenal mucus gel
in vivo can also retard access to the mucosa of relatively small
molecules and, furthermore, strongly suggests that such a barrier can
be maintained by the gel layer at the duodenal surface despite luminal
perfusion with fluid. If there are discontinuities in the mucus layer,
they seem few and small. Further studies are necessary to distinguish
whether the barrier effect of mucus against low-dose
PGE2 is a function of its
retarding diffusion per se or whether it is because of binding of the
prostaglandin to the mucin or other components of the mucus gel.
To avoid cellular damage, the concentration of papain in the luminal perfusate and time of exposure were kept at the minimum necessary to remove most of the mucus layer, and it should be emphasized that the papain-perfused duodenal mucosa showed clear (and enhanced) responses to the various secretagogues tested in the present study. The morphological examination of fixed sections of the duodenum provides further strong evidence that treatment with papain, as used in the present study, does not damage the mucosa. The absence of an effect of papain on the blood-to-lumen clearance of 51Cr-EDTA should also be noted. The latter provides another reliable estimate of mucosal integrity (25).
In the stomach, the adherent mucus gel over the surface of the mucosa
appears firmer (1, 20, 32) than that in the duodenum (29). The barrier
to migration of peptide macromolecules of the gastric mucus layer in
vivo has been exemplified in humans by the uptake by the gastric mucosa
of cationized ferritin only after removal of the mucus layer (35). In
the rat, damage from excess luminal pepsin (molecular mass of 34.7 kDa)
occurred only when the enzyme itself had digested away the adherent
gastric mucus gel layer over a period of hours (4). Further studies seem necessary for an evaluation of a possible difference between the
stomach and duodenum in mucus gel continuity and permeability. The
greater gross surface area in the duodenum together with the inherent
capability of the duodenum to secrete
HCO3 at high rates (10, 11) may make
this tissue particularly suitable for the present approach.
Cholera toxin is a well-established stimulant of NaCl and water
secretion in the more distal small intestine, and it has been demonstrated that cholera toxin-specific receptors on the enterocytes (22), as well as reflexes within the enteric nervous system involving
nicotonic transmission (19), mediate the secretory response. The onset
of action is, however, often remarkably slow and continues after
removal of the toxin (19). Our experiments offer one explanation for
this in terms of the barrier effect of the mucus layer. Thus removal of
the mucus layer with papain increased both rapidity of onset and
magnitude of stimulation of cholera toxin-induced
HCO3 secretion (Fig. 1). Pretreatment
with the nicotinic antagonist hexamethonium did not affect the response
to cholera toxin, strongly suggesting that the stimulation of the
duodenal secretion reflects an action on enterocyte receptors and thus
migration of the (cholera toxin) probe macromolecule to the epithelial surface.
Glucagon and PGE2, like cholera
toxin used as probe molecules in the present study, are
well-established stimulants of duodenal mucosal
HCO3 secretion (10, 11).
PGE2 has been shown to increase in
intracellular cAMP production in a mixture of guinea pig duodenal crypt
and villus enterocytes (28), but the cell type responding to glucagon
(villus or crypt) and the mode of intracellular signaling have not been
clarified. However, villus as well as crypt duodenal enterocytes
respond to vasoactive intestinal polypeptide and dopamine with an
increase in intracellular cAMP production (30) and to carbachol and
cholecystokinin octapeptide with a rise in intracellular
Ca2+ (6). Migration of luminally
instilled glucagon and PGE2 across the mucus gel to the lumen-facing villus tip region may be sufficient for eliciting an HCO
3 secretory response.
The HCO3 secretion and in particular
the ability of duodenal mucosa to respond to luminal acid with a rise in HCO
3 secretion protect acid-exposed
mucosa in animals (10), and the HCO
3
secretion is decreased in patients with acute and chronic duodenal
ulcer disease (17, 24). Interestingly, it was recently reported that
eradication of H. pylori infection in
part restores the HCO
3 secretion in
such patients (16). Acute exposure to water extracts containing
H. pylori cytotoxin, however, did not
inhibit but caused a small increase in duodenal
HCO
3 secretion in the present study.
This occurred only after removal of the mucus layer by papain and was
not seen with extracts from an isogenic mutant (VacA
) not
producing cytotoxin. The cytotoxin did not affect mucosal permeability
when measured as 51Cr-EDTA
clearance. These combined results would suggest the toxin acts as a
(modest) stimulant, rather than as an inhibitor of transcellular transport of HCO
3. Further studies are
required for an evaluation of the HCO
3
secretory response to H. pylori
cytotoxin, but it should be noted that the stimulation of secretion by
cholera toxin observed in the present study very probably reflects an
action mediated by enterocyte receptors at the epithelial surface. It
is possible that inhibition of secretion reflects effects of
H. pylori toxins and/or the process of
chronic inflammation on neurohumoral pathways (9, 10, 14, 16) mediating
the physiological control of the HCO
3 secretion.
In summary, this study shows that the adherent duodenal mucus gel layer
can significantly hinder mucosal access to even small-sized luminal
HCO3 secretagogues and severely limit the access of larger-sized toxins. Furthermore, the release of H. pylori cytotoxin and other
bacteriotoxins at the cell-mucus interface may thus greatly enhance
their effects on mucus-covered gastrointestinal mucosa. The useful
experimental model described here also emphasizes that access through
the mucus is an important consideration when studying the effects of
luminal secretagogues and other agents on the underlying epithelium.
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ACKNOWLEDGEMENTS |
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We thank Hjördis Andersson for skillful technical assistance.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (grants 3515 and 10848).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Flemström, Dept. of Physiology, Uppsala Univ. Biomedical Center, PO Box 572, SE-751 23 Uppsala, Sweden (E-mail: Gunnar.Flemstrom{at}Fysiologi.uu.se).
Received 31 December 1998; accepted in final form 6 May 1999.
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