Department of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
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ABSTRACT |
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The present study was conducted to elucidate the mechanisms by which Helicobacter pylori (HP)-derived ammonia causes gastric mucosal injury. Intact sheets of guinea pig gastric fundic mucosae were incubated in Ussing chambers. Both the luminal and the serosal pH were kept at 7.4. Transmucosal potential difference (PD) and electrical resistance (R) were monitored as indices of mucosal integrity. Restitution was evaluated by recovery of PD, R, and transmucosal [3H]mannitol flux after Triton X-100-induced mucosal injury. The effects of luminal or serosal NH4Cl on function and morphology of uninjured or injured mucosae were examined. In uninjured mucosae, serosal NH4Cl induced more profound decreases in PD and R and more prominent vacuolation in gastric epithelial cells than did luminal NH4Cl. In contrast, luminal NH4Cl markedly inhibited restitution in injured mucosae and caused an extensive vacuolation in gastric epithelial cells, as did serosal NH4Cl. Transmucosal ammonia flux was greater in the injured than in the uninjured mucosae. These results suggest that 1) basolateral membrane of gastric epithelial cells is more permeable to ammonia than apical membrane and 2) luminal ammonia, at concentrations detected in HP-infected gastric lumen, retards restitution in injured mucosae.
Helicobacter pylori; vacuolation; apical membrane; basolateral membrane; epithelial cells
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INTRODUCTION |
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IT HAS BEEN SUGGESTED THAT chronic gastric mucosal infection with Helicobacter pylori (HP) is closely associated with development of gastritis, peptic ulcers, and possibly with gastric carcinoma (2, 10, 14, 36). HP has been shown to possess strong urease activity, which produces a high concentration of ammonia within the gastric lumen as a result of hydrolysis of urea in gastric juice (15, 23).
Ammonia has been shown to exist as two different forms, NH3 and NH4+. It has been reported that NH3, a small lipophilic molecule (molecular weight 17.0), is readily permeable across the phospholipid bilayer of cell membranes in a variety of tissues (19). In contrast, NH4+, a monovalent cation, does not diffuse across the cell membrane but does pass through cation channels that are located on the basolateral membrane of the gastric epithelial cells (8, 13). Since ammonia has a pKa of 9.0, most of the ammonia exists as NH4+ under physiological conditions. In this study, the sum of NH3 concentration ([NH3]) and NH4+ concentration ([NH4+]) is hereafter referred to as ammonia.
A number of studies have suggested that ammonia plays an important role in the development of gastric mucosal injury (15, 25, 31, 35, 38). For example, ammonia at concentrations produced by HP has been shown to cause injury in isolated human gastric epithelial cells in vitro (31) and in rat gastric mucosa in vivo (25, 35). Ammonia has been shown to increase H+ permeability across the gastric mucus gel layer (9). We have previously shown that luminal ammonia at neutral pH enhances H+ backdiffusion across intact sheets of bullfrog gastric mucosa in vitro (42). Ammonia has been shown to enhance formation of cytoplasmic vacuolation induced by HP-derived vacuolating cytotoxin (Vac A protein) in vitro (6). Ammonia in HP-infected stomach has also been shown to react with free radicals generated by intramucosal neutrophils, which results in production of an extremely toxic substance, monochloramine, thereby causing severe mucosal injury (7, 15, 17, 26, 34).
Some investigators, however, have pointed out that luminal ammonia
alone, especially at concentrations detected in HP-infected subjects,
does not cause injury in normal uninjured gastric mucosa (7, 17, 38). On the basis of
these results, these investigators concluded that ammonia does not play
a central role in the pathogenesis of gastric mucosal injury in
HP-infected gastric mucosa in vivo. In contrast, we have previously
shown that ammonia, when present at the serosal side of the mucosa,
even at much lower concentrations than that detected in HP-infected
stomachs, strongly inhibits both H+ secretion and
Cl transport across the gastric mucosa (42).
On the basis of these findings, we hypothesized that luminal ammonia in
HP-infected gastric mucosa may diffuse into the basolateral side
through the injured apical surface and impair function of the gastric
epithelial cells from the basolateral side, ultimately resulting in
extensive mucosal injury.
In the present study, we demonstrate that 1) the basolateral membrane of gastric epithelial cells is more permeable to ammonia than the apical membrane and 2) luminal ammonia, at concentrations detected in an HP-infected subject, does not cause injury in normal uninjured mucosa but does retard restitution of injured gastric mucosa.
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METHODS |
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Animals
Female guinea pigs (250-300 g, Hartley strain) were killed under anesthesia induced by inhalation of diethyl ether. The stomachs were removed and divided into paired halves by incising the greater and lesser curvatures of the fundus. The muscularis propria was stripped from the mucosa with small scissors and fine forceps under microscopic control.Electrophysiology
Intact sheets of resting guinea pig gastric fundic mucosae were mounted in Lucite Ussing chambers in vitro. The chambers were connected to water-jacketed gas-lift reservoirs, which were attached to keep the temperature of the mucosae constant at 37.0°C. The serosal side was bathed with a Ringer solution, buffered with 25 mM HEPES, pH 7.4 (see Solutions), and gassed with 100% O2. The luminal side was initially bathed with 150 mM NaCl and gassed with 100% O2 (pH ~6.0-7.0). H+ secretion was inhibited by treatment of the serosal side with 0.1 mM omeprazole for 60 min. After removing omeprazole from the serosal solution, both sides of the tissue were bathed with a fresh HEPES-Ringer solution. Then the tissue was allowed to settle for another 60-min period. Transmucosal potential difference (PD) and electrical resistance (R) were monitored throughout the experiments. We confirmed that PD and R were maintained at reasonable values for 3-4 h, as long as the mucosae were kept at the initial control conditions. R was calculated from the change in PD induced by passing 100 µA of electrical current from the luminal to the serosal side for 0.5 s every 2 min. The mean initial values of PD and R in uninjured control mucosae wereMorphology
Gastric mucosal specimens for electron and light microscopy were fixed immediately after the electrophysiological experiments by immersion in 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.02% trinitrophenol (picric acid) in 0.1 M cacodylate buffer for at least 2 h at room temperature. Tissues were then postfixed with 1% osmium tetroxide (OsO4) in 0.1 M cacodylate buffer for 1 h, treated with 1% uranyl acetate in maleate buffer (pH 5.2), and embedded in an Epon-Araldite mixture. Embedded tissues were sectioned at ~0.8-1.0 µm. The semithin sections were stained with toluidine blue and were examined by light microscopy. Some of the blocks were further sectioned at ~0.07-0.09 µm. The ultrathin sections were stained with uranyl acetate and lead citrate for transmission electron microscopy. Five slides for light microscopy and five sections for electron microscopy were prepared from each group. Those samples were coded and were evaluated in a blind fashion by two investigators (H. Suzuki and A. Yanaka).Cytoplasmic vacuolation was assessed by electron microscopy. The degree of vacuolation was classified according to the following criteria. When vacuolation was found in >90% of the gastric epithelial cells (i.e., the gastric gland cells and the surface mucous cells), the degree of vacuolation was referred to as "severe." When vacuolation was noted in ~50-90% of the cells, the degree was referred to as "moderate." When vacuolation was noted in <50% of the cells, the degree was referred to as "mild."
Restitution was evaluated by light microscopy. Degree of restitution was classified as either "complete" or "incomplete." We referred to complete restitution only when the lamina propria was perfectly covered with epithelial cells (i.e., epithelial continuity is established) in all areas of the five samples we evaluated. In contrast, we referred to incomplete restitution when even a single focus of absence of epithelial continuity was found in any of the five slides we examined.
[3H]Mannitol Flux Study
To assess mucosal permeability, [3H]mannitol flux (MF) from the serosal to the luminal solution was measured under short-circuited conditions, as described by Moore et al. (24). In brief, [3H]mannitol, 5 µCi, was placed in the serosal solution. One-milliliter aliquots were collected from the luminal bath at 15-min intervals throughout the experiments. The entire luminal solution was replaced with well-oxygenated fresh luminal solutions at each 15-min interval immediately after the 1-ml aliquot was collected. Unlabeled mannitol (5 mM) was present in both the serosal and the luminal solutions throughout the experiment to eliminate a solute gradient between the two sides. MF at each 15-min period was evaluated from the radioactivity of 3H detected in each sample.Experimental Protocol for Restitution Study
To cause superficial mucosal injury, the luminal side was exposed to 2% Triton X-100 for 5 min, as described previously (24). This treatment induced rapid and marked decreases in PD and R, accompanied by a reciprocal increase in MF. Morphology of the Triton X-100-treated mucosae showed superficial injury of gastric mucosa (see RESULTS). After the luminal solution was replaced with fresh HEPES-buffered Ringer solution, the tissues were incubated for 2 h, during which PD, R, and MF showed gradual recovery. Restitution was quantitatively evaluated by the recovery of PD and R and/or the changes in MF after the injury, as described in our previous report (41) and in the report by Moore et al. (24). Restitution was also evaluated morphologically by light microscopy of the mucosae fixed at the end of the experiment.Measurement of Ammonia Flux From the Luminal to the Serosal Side
To examine the mucosal permeability of ammonia, 10 mM NH4Cl was added to the luminal solution of the uninjured or the injured mucosae. In injured mucosae, NH4Cl was added immediately after removal of Triton X-100 from the luminal solution. One-milliliter aliquots of the serosal solution were collected every 30 min after addition of the luminal NH4Cl. Concentrations of NH3 in each sample were determined using a kit from Sigma (St. Louis, MO), which is based on reductive amination of 2-oxoglutarate in the presence of glutamate dehydrogenase, as described elsewhere (27). Total ammonia concentration in the serosal solution ([NH4+ + NH3]) was calculated from the serosal [NH3], taking into account that NH3:NH4+ is 1:40 at pH 7.4, since pKa of ammonia has been shown to be 9.0 (20). Transmucosal flux of ammonia from the luminal to the serosal side was estimated from the values of serosal [NH4+ + NH3]. Ammonia flux was expressed as the percentage of the total ammonia added to the luminal solution.Chemicals
Omeprazole was kindly provided by Astra (Hässel, Mölndol, Sweden). All other chemicals were purchased from Wako Pure Chemical (Osaka, Japan).Solutions
The serosal solution contained (in mM) 122 Na+, 5.0 K+, 1.8 Ca2+, 0.8 Mg2+, 130 ClStatistical Analysis
Student's paired or unpaired t-tests were used for the comparison of paired or unpaired values, respectively. P < 0.05 was considered significant. Results were expressed as means ± SE. ![]() |
RESULTS |
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Effects of Luminal or Serosal NH4Cl on Uninjured Gastric Mucosae
This series of experiments was conducted to determine whether the serosal side of the uninjured normal mucosa is more susceptible to the action of NH4Cl than the luminal side. Both the luminal and the serosal side were bathed with the HEPES-Ringer solution (pH 7.4) throughout the experiment.Changes in PD and R after addition of luminal or serosal
NH4Cl to uninjured control mucosae.
Addition of 30 mM NH4Cl either to the luminal or the
serosal side for 2 h significantly decreased PD and R
(Fig. 1). The magnitudes of the decreases
in PD and R induced by the serosal NH4Cl were significantly greater than those induced by the luminal
NH4Cl (Fig. 1)
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Dose-response studies on the effect of luminal or serosal
NH4Cl/NaCl on PD and R.
In this series of experiments, changes in PD and R were
examined at 30 min after addition of graded doses of NH4Cl
or NaCl (1, 3, 10, 30, and 100 mM) to either the serosal or the luminal side. Addition of NaCl, at concentrations of ~1-30 mM, to either the serosal or the luminal side did not affect PD or R.
However, 100 mM NaCl in either the luminal or the serosal solution
caused significant decreases in PD and R, although the
magnitudes of the decreases in PD and R were significantly
less than those induced by 100 mM NH4Cl (Fig.
2).
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Effect of luminal or serosal NH4Cl on morphology.
Morphological examination by light and electron microscopy demonstrated
that the mucosae treated with 30 mM NH4Cl in the luminal solution for 2 h showed a mild degree of vacuolation, mainly in the gastric glands located toward the basolateral side of the mucosa
(Fig. 3, A and B).
In contrast, the mucosae treated with 30 mM NH4Cl in the
serosal solution showed severe degree of vacuolation, characterized by
an extensive vacuolation of almost all the cells in both the gastric
glands and the surface mucous cells (Fig. 3C). Low-power
electron micrographs showed numerous small vacuoles in the surface
mucous cells and large vacuoles in oxyntic cells in gastric glands
(Fig. 3D).
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Effects of inhibition of the basolateral cation channels on the
changes in PD and R induced by serosal NH4Cl.
This series of experiments was conducted to examine the possibility
that the entry of NH4+ into the gastric epithelial
cells occurred via the basolateral K+ channels and the
Na+-K+-2Cl cotransporters, since
this has been reported in renal epithelial cells (3,
20, 21) and in salivary acinar cells
(12). The serosal side of the mucosa was exposed to
various concentrations of either BaCl2 (0.1, 0.3, or 1 mM)
or bumetanide (0.01, 0.03, or 0.1 mM), which have been shown to block
the K+ channel and the
Na+-K+-2Cl
cotransporter,
respectively (21). Addition of BaCl2 to the serosal side induced a rapid decrease in PD and an increase in R (Fig. 4, A and
B). Serosal bumetanide also caused a rapid decrease in PD
but did not alter R (Fig. 4, A and B).
PD and R reached stable values during the 30-min period
after addition of these agents. Subsequently, 30 mM NH4Cl
was added to the serosal side. Serosal BaCl2, at
concentrations approximately between 0.1 and 1 mM, dose-dependently
attenuated the decreases in PD and induced by serosal NH4Cl
(Fig. 4, C and D). Serosal bumetanide, at
concentrations approximately between 0.01 and 0.1 mM, also
dose-dependently mitigated the NH4Cl-induced decreases in
PD and R (Fig. 4, E and F).
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Effects of NH4Cl on Restitution in Triton X-100-Treated Injured Mucosae
This series of experiments was conducted to determine whether luminal or serosal NH4Cl, at physiological concentrations, could inhibit restitution. Therefore, restitution was evaluated in the presence or absence of 30 mM NH4Cl in either the luminal or the serosal side. In other tissues, 30 mM NaCl, instead of 30 mM NH4Cl, was added to either the luminal or the serosal side to exclude the possibility that increases in osmolarity affect restitution.Effects of luminal or serosal NH4Cl on
electrophysiology and MF after injury.
In the absence of 30 mM NH4Cl, PD and R
gradually increased, whereas MF rapidly decreased during a recovery
period after the Triton X-100-induced injury (Fig.
5, A-C). In
contrast, the recovery of PD, R, and MF was markedly
inhibited by either the luminal or the serosal NH4Cl (Fig.
5, A-C). There was no evidence of recovery of PD, R, or
MF at 2 h after the injury in tissues exposed to NH4Cl
(Fig. 5, A-C).
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Effects of luminal or serosal NaCl on electrophysiology and MF after injury. This series of experiments was conducted to examine the possibility that an increase in osmolarity affects restitution. Restitution was examined after addition of 30 mM NaCl, instead of 30 mM NH4Cl, to either the luminal or the serosal side. Addition of 30 mM NaCl to either the luminal or the serosal side did not affect either the recovery of PD and R or the decrease in MF after injury (Fig. 5, D-F).
Effects of luminal or serosal NH4Cl on morphology of
injured mucosae.
Exposure of the luminal surface to 2% Triton X-100 for 5 min induced
superficial injury of the gastric mucosa, characterized by exfoliation
of large amounts of the surface mucous cells without causing marked
injury of the gastric glands (Fig.
6A). In the absence of
NH4Cl, the denuded surface of the injured mucosae was almost completely covered with the surface mucous cells at 2 h after the injury (Fig. 6B).
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Roles of basolateral K+ channels in restitution.
In this series of experiments, we tested the hypothesis that inhibition
of restitution by NH4Cl is caused by blockade of the basolateral K+ channels by NH4+ in gastric
epithelial cells, since recent studies have suggested the importance of
the basolateral K+ channels in cell migration
(28, 29). We therefore examined the effects
of inhibition of basolateral K+ channels by serosal 1 mM
BaCl2 on restitution in Triton X-100-injured gastric
mucosae. Serosal BaCl2 markedly prevented recovery of R (Fig. 7A), and
retarded the decrease in MF after injury (Fig. 7B),
indicating that the basolateral K+ channels play an
important role in restitution of injured guinea pig gastric mucosae.
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Ammonia Flux from the Luminal to the Serosal Side
In both the normal uninjured and the injured mucosae, [NH4+ + NH3] in the serosal solution gradually increased over time (Fig. 8A). The [NH4+ + NH3] detected in the serosal solution in the injured mucosa was significantly greater than that detected in the uninjured control mucosa (Fig. 8A). The ammonia flux from the luminal to the serosal side during the initial 30-min period was 0.08 ± 0.01% and 1.15 ± 0.11% in the uninjured and the injured mucosae, respectively (Fig. 8B).
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DISCUSSION |
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The present study clearly demonstrates that the serosal surface of uninjured normal guinea pig gastric fundic mucosa is more sensitive to the action of NH4Cl than the luminal surface. There are several possible explanations for the difference in NH4Cl permeability between the luminal and the serosal membranes.
First, the difference could be attributable to asymmetrical
distribution of the cation channels in highly polarized gastric epithelial cells (8, 13). Because ammonia has
a pKa of 9.0 (20), calculated
[NH4+]:[NH3] at pH 7.4 is ~40:1. The
greater sensitivity of the serosal side of the mucosa to ammonia could
be attributable to the large NH4+ permeability of the
basolateral membrane of the gastric epithelial cells. As stated in the
introduction, NH4+ does not diffuse through the
phospholipid bilayer of the cell membrane but does pass through several
kinds of cation channels. A number of recent studies in renal
epithelial cells and in salivary acinar cells have clearly shown that
NH4+ acts as a surrogate for K+ and passes
through both the K+ channels (3,
21) and the
Na+-K+-2Cl cotransporters
(12, 20, 21), although this has
not been examined previously in gastric epithelial cells
(8, 13). Since the existence of these cation
channels has been demonstrated on the basolateral side of the gastric
epithelial cells (8, 32, 33), it
seems reasonable to propose that NH4+ passes through
both the K+ channel and the
Na+-K+-2Cl
cotransporter on the
basolateral surface of gastric epithelial cells, thereby rendering the
basolateral membrane more permeable to NH4Cl than the
apical membrane. This possibility is strongly supported by the present
results that the decrease in PD and R induced by the serosal
NH4Cl was dose-dependently inhibited by serosal
BaCl2 or by serosal bumetanide (Fig. 4), agents known to
block the K+ channel and the
Na+-K+-2Cl
cotransporters,
respectively (21). Furthermore, our morphological findings
that serosal NH4Cl caused more prominent cytoplasmic vacuolation in gastric glands than did luminal NH4Cl also
support this proposal. Thus the present results strongly suggest that the serosal membrane of well-polarized gastric epithelial cells is more
permeable to NH4+ than the apical membrane. The
significant difference in ammonia permeability of the serosal and the
luminal membranes has also been demonstrated by other studies in
isolated gastric glands (39) and in T84 cell monolayers
(27).
Second, the difference in NH4Cl permeability could be attributable to the difference in the permeability of NH3 between the luminal and the serosal membranes of gastric epithelial cells, since recent studies have suggested the relatively impermeable nature of the apical membrane to NH3 in well-polarized epithelial cells (18, 30).
Third, it is also conceivable that the basolateral
Cl/HCO3
exchangers in oxyntic cells
raise ambient pH on the basolateral surface of the mucosa by extruding
cytoplasmic HCO3
(i.e., an alkaline tide)
(1, 22), thereby enhancing conversion of
NH4+ to NH3. The NH3 could then
diffuse directly into the cells through the cell membrane. This
possibility is supported by the present finding that luminal
NH4Cl induced more prominent vacuolation in gastric glands
than in the surface mucous cells. It is also conceivable that oxyntic
cells and/or peptic cells may be more susceptible to ammonia in terms
of vacuolation than the surface mucous cells, even though we do not
have direct evidence for this possibility. The present results also
suggest that vacuolation, per se, is not related to cell death, since
we did not see necrotic changes in the vacuolated cells. Our findings
are in concert with the studies on the effect of ammonia on morphology
of isolated rabbit gastric glands in vitro (16).
The present study shows that the luminal surface of the Triton X-100-treated injured mucosa is more vulnerable to NH4Cl than is the normal uninjured mucosa. The increased vulnerability of the luminal surface to ammonia after injury could be attributable to the increased ammonia permeability as a result of the mucosal injury caused by Triton X-100, since the ammonia flux into the serosal solution showed a >10-fold increase after the injury. Recent studies have shown that HP disrupts tight junctions, thereby increasing paracellular permeability, which could also facilitate paracellular diffusion of ammonia from the luminal to the serosal side (5, 11). Thus, in HP-infected gastric mucosa in vivo, significant amounts of luminal ammonia could diffuse across the mucosa paracellularly through the injured sites and enter the cell from the basolateral membrane of the epithelial cells, thereby inhibiting restitution. In addition, it is also possible that the serosal ammonia reacts with the free radicals produced by intramucosal neutrophils and produces monochloramine (NH2Cl), which has been shown to cause more severe damage in the epithelial cells than does ammonia alone (7, 15, 17, 26, 34).
The present study shows that relatively low concentrations of ammonia, ~10-30 mM NH4Cl at pH 7.4, which are almost equivalent to those detected in the gastric lumen in the HP-infected subjects (37), strongly inhibit restitution. We assume that the luminal surface of gastric epithelial cells in HP-infected subjects is exposed to higher concentrations of ammonia than that in gastric juice, since the majority of HP live within the adherent mucus gel layer. In contrast, in the present experimental system, [NH4+ + NH3] at the luminal surface of the mucosae is presumably not greater than that in the luminal solution, since we added NH4Cl to the luminal solution and HP was not present within the mucus gel layer. Together, we believe that the [NH4Cl], ~10-30 mM at pH 7.4, used in the present study are clinically relevant concentrations.
The mechanisms by which NH4Cl causes gastric mucosal injury
and retards restitution have not been well clarified. It has been suggested that ammonia inhibits mitochondrial respiration and subsequent energy metabolism (38). We have previously
shown that hypoxia inhibits restitution in bullfrog gastric mucosa in vitro (40). Thus it seems reasonable to assume that
ammonia abolishes reorganization of actin filaments of the epithelial cells, which is an energy-dependent process, and is essential for cell
migration on the denuded surface of the injured mucosae (4). Another possibility is that inhibition of the
basolateral K+ channels of gastric epithelial cells by
ammonia may contribute to retardation of restitution. This possibility
is supported by the present results, which showed that restitution is
markedly prevented by blockade of the basolateral K+
channels by serosal BaCl2 (Fig. 7). Recent studies
in renal epithelial cells have suggested that basolateral
K+ channels play an important role in migration of the
cells (28, 29). Furthermore, we have
previously shown that NH4+ inhibits electrogenic
Cl transport across the bullfrog gastric mucosa by
blocking the basolateral K+ channels (42).
Inhibition of the basolateral K+ channels by ammonia has
also been reported by studies in T84 cell monolayers (27).
Thus it seems likely that inhibition of restitution by ammonia is, at
least in part, related to inhibition of the basolateral K+
channels by ammonia.
In the present study, we evaluated restitution by the recovery of both
the transmucosal electrical resistance and the transmucosal flux of
3H-labeled mannitol, since both of these parameters have
been shown to correlate with morphological restitution (3,
20). Each of these methods, however, has some
disadvantages. For example, the electrical resistance is affected by
various factors, such as changes in H+ secretion of the
mucosae or by changes in concentration of ambient Na+,
K+, or Cl. The value of the MF is not
affected by those factors, although this technique takes more time and
is more expensive than measurement of the electrical resistance. For
these reasons, we have used both measurements to enhance the accuracy
of assessing restitution.
With regard to changes in osmolarity on electrophysiological parameters, we have found that an increase in osmolarity from 300 to 500 mosM by the addition of 100 mM NaCl decreases the value of PD and R in uninjured mucosae. However, we have also confirmed that an increase in osmolarity by 60 mosM by the addition of 30 mM NaCl did not affect either the electrophysiological parameters in uninjured mucosae or the restitution after injury. These results indicate that the decreases in PD and R and the retardation of restitution after addition of 30 mM NH4Cl were not induced by increases in osmolarity but rather were caused by the effects of NH4+ or NH3 on the physiology of gastric epithelial cells.
In summary, the present study suggests that the serosal surface of uninjured normal guinea pig gastric mucosa is more susceptible to the effects of ammonia than the apical surface and that low concentrations of luminal ammonia, at physiological concentrations, prominently retard restitution in injured mucosae.
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ACKNOWLEDGEMENTS |
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We gratefully thank Dr. William Silen (Department of Surgery, Beth Israel Hospital, Harvard Medical School) and Dr. Susumu Ito (Department of Neurobiology, Harvard Medical School) for critical review of the manuscript. We also thank Noriko Sugae for excellent technical assistance with the electron microscopy.
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FOOTNOTES |
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This work was supported by Grant-in-Aid for Scientific Research no. 10670450 from the Ministry of Education, Science, Sports, and Culture of Japan.
Address for reprint requests and other correspondence: A. Yanaka, Dept. of Gastroenterology, Institute of Clinical Medicine, Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan (E-mail: ynk-aki{at}md.tsukuba.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 29 July 1999; accepted in final form 29 January 2000.
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