Department of Physiology, Biomedical Center, Uppsala University, S-751 23 Uppsala, Sweden
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ABSTRACT |
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The acid-secreting gastric mucosa
resists intraluminal acid better than the nonsecreting. Here we
investigated pH at the epithelial cell surface, mucosal permeability,
and blood flow during intraluminal administration of acid (100 mM) in
acid-stimulated and nonstimulated gastric corpus mucosae. Surface
pH (H+-selective microelectrodes), permeability (clearance
of 51Cr-EDTA), and mucosal blood flow (laser-Doppler
flowmetry) were studied in Inactin-anesthetized rats. Acid secretion
was stimulated with pentagastrin (40 µg · kg1 · h
1) or
impromidine (500 µg · kg
1 · h
1), or
HCO3
(5 mmol · kg
1 · h
1) given
intravenously. Surface pH was only slightly reduced by intraluminal
acid in acid secretion-stimulated or HCO3
-treated
rats but was substantially lowered in nonstimulated rats. Clearance
increased threefold and blood flow increased by
75% in
nonstimulated rats. During stimulated acid secretion or intravenous infusion of HCO3
, clearance was unchanged and blood
flow increased by only
30% during intraluminal acid. Increased
epithelial transport of HCO3
buffering the mucus gel
is most probably the explanation for the acid-secreting mucosa being
less vulnerable to intraluminal acid than the nonsecreting.
gastric acid; laser-Doppler flowmetry; permeability; rat
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INTRODUCTION |
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THE GASTRIC MUCOSA IS FREQUENTLY exposed to HCl in high concentrations. Despite this exposure, acid is prevented from entering and damaging the mucosa. Thus the gastric mucosa must possess a functional barrier against the acid in the lumen.
We have shown previously that a pH gradient is maintained in the mucus covering the corpus mucosa down to a luminal pH of 2 (31). Thus a pH of 7.2 was found at the epithelial cell surface in stomachs stimulated to produce acid and a pH of 6.9 in nonsecreting stomachs, whereas the luminal pH was 2. Earlier studies performed by others (29, 30) have also shown that a pH gradient is maintained in the mucus gel in the nonstimulated stomach down to a luminal pH of 2-3. The gradient is destroyed, however, at a pH of 1.4 (and lower) in the lumen in the nonstimulated stomach (29, 30), but to our knowledge this has not been tested in the acid-secreting stomach in vivo.
For each H+ produced by the parietal cells and released
into the lumen in the acid-secreting stomach, one
HCO3 is formed and transported into the blood
(34). The HCO3
probably reaches the
surface epithelium through the microcirculation (5, 6).
The question arises as to whether the HCO3
transport
from interstitial and endogenous sources is efficient in maintaining a
neutral pH at the cell surface of the gastric corpus mucosa during
topical administration of strong acid, i.e., pH 1 in the lumen. In this
study, we therefore investigated the epithelial cell surface pH, the
mucosal permeability, and the mucosal blood flow during topical
administration of 100 mM HCl in rats stimulated to produce acid either
with pentagastrin or with the H2 receptor agonist
impromidine as well as in nonstimulated rats that only had a low
spontaneous acid secretion. In other experiments, to test whether
blood-delivered HCO3
alters the mucosal response to
topical administration of acid in the nonstimulated mucosa, we
administered NaHCO3 intravenously in rats.
For studying the gastric corpus mucosae, we used an in vivo model whereby several variables can be followed continuously. The epithelial cell surface pH was measured with H+-selective microelectrodes, and the mucosal permeability was assessed by measuring the clearance of 51Cr-EDTA from blood to lumen. Mucosal blood flow was measured by laser-Doppler flowmetry (LDF).
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MATERIALS AND METHODS |
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Animal Preparation
Male Sprague-Dawley rats (Möllegaard Breeding Center, Ejby, Denmark), weighing 150-250 g, were kept under standardized conditions of temperature (21-22°C) and illumination (12:12-h light/darkness). They were allowed to adjust to this environment in cages with mesh bottoms for at least 4 days before the experiments began, with free access to tap water and pelleted food (Ewos, Södertälje, Sweden). The rats were deprived of food for 18-20 h before the experiments but had free access to water right up to the beginning of the experiment. They were anesthetized with 120 mg/kg body wt of 5-ethyl-5-(1-methylpropyl)-2-thiobutabarbital sodium (Inactin) injected intraperitoneally. Tracheotomy was performed to facilitate spontaneous breathing, and body temperature was maintained at 37.5 ± 0.5°C by means of a heating pad controlled by an intrarectal thermistor. For blood withdrawal, a PE-90 cannula, containing heparin dissolved in saline (100 IU/ml), was inserted into the left common carotid artery. The right femoral artery was cannulated for continuous recording of arterial blood pressure, and the right femoral vein was cannulated for continuous infusion of 1.0 ml/h of Ringer solution containing, in mM: 25 NaHCO3, 120 NaCl, 2.5 KCl, and 0.75 CaCl2. The left femoral vein was cannulated when needed for drug infusion. The preparation of the gastric mucosa for intravital microscopy has been described previously (12). Briefly, exteriorization of the mucosa through a midline abdominal incision was followed by an incision along the greater curvature in the forestomach. The rat was placed on a Lucite table with a part of the corpus of the stomach loosely draped over a truncated cone in the center of the table, with the mucosal surface facing upwards. A double-bottom "mucosal chamber" with a hole in the bottom was fitted over the mucosa, exposing ~1.2 cm2 of the mucosa through the hole, and the junction was sealed with silicone grease. The mucosal chamber did not touch the mucosa, so as not to impair blood flow. The chamber was filled with 5 ml of unbuffered 0.9% saline, kept at 37°C by perfusing the double-bottom chamber with warm water. The saline was replaced at constant intervals of 15 min and titrated (Autobürette ABU 91; Radiometer, Copenhagen, Denmark) with 10Epithelial Cell Surface pH
The H+ concentration in the mucus gel at the epithelial cell surface was measured with H+-selective microelectrodes. Glass tubing (borosilicate tubing with omega dot, OD 1.2 mm, ID 0.9 mm; Frederik Haer, Brunswick, ME) was pulled with a pipette puller (pp-83; Narishige Scientific Instrument Laboratory, Tokyo, Japan) to a tip diameter of 1-3 µm. These pipettes were siliconized at 200°C with tributylchlorosilane according to the procedure described by Tsien and Rink (35) and stored for up to 1 wk at 100°C. Before each experiment, the electrodes were filled up to a distance of 330-550 µm from the tip with a proton cocktail (H+ Ionophore II-Cocktail). The remainder of the electrode was filled with HEPES buffer at pH 7.4, connected by an Ag-AgCl wire to a dual-differential electrometer with a high input impedance (FD 223; Biomedical Center, Uppsala, Sweden) and held in a pipette holder (MEH3SF 1.2; Mark Finlay, WPI, Aston, England). The reference electrode, filled with 3 M KCl and connected by an Ag-AgCl wire to the ground of the electrometer, was inserted into another holder (MEH3SF 3.0; Mark Finlay) so that the tip dipped into the isotonic NaCl solution covering the surface of the mucosa. To eliminate electric disturbances, the experiments were performed in a Faraday cage. The electrodes were calibrated before and after each experiment in solutions at 37°C. The solutions were made isosmolar (300 mosM) with NaCl. Solutions with pH values of 1-3 were obtained by the addition of HCl (155 mM) to an unbuffered NaCl solution (155 mM), and those in the pH range of 4-8 were obtained by the addition of HCl or NaOH to a solution containing 10 mM HEPES and 140 mM NaCl. The electrode was inserted into the gel from the luminal solution at an angle of 30-35° to the surface with the aid of a micromanipulator (Leitz, Wetzlar, Germany). Graphite particles (activated charcoal, extra pure; Merck, Darmstadt, Germany) were placed on the mucus gel to visualize the position of the luminal surface. The cell surface was usually visible through the microscope. In some rats, mostly those with thicker mucus layers, the surface of the cells was not clearly visible. In those cases, the mucosa could be seen to be displaced sidewise when the electrode touched the cell surface. When this occurred, the electrode was withdrawn until the mucosa no longer appeared displaced. A "digimatic indicator" (IDC Series 543; Mitutoyo, Tokyo, Japan) was connected to the micromanipulator for measurement of the distance (D) of movement of the electrode from the mucus gel surface to the luminal cell surface. The distance in the vertical direction (90° to the surface) from the luminal surface of the mucus gel to the cell surface [mucus gel thickness (T)] was calculated from the angle (30-35°) of the insertion and the distance of the movement of the electrode described above. For this calculation we used the formula: T = D × sine angle (30-35°).Mucosal Permeability
Mucosal permeability was determined by measuring the clearance of 51Cr-EDTA from blood to lumen (25). The technique appears to provide a highly reproducible measure of mucosal integrity and has the advantage that each animal can serve as its own control (2, 4, 9). After completion of surgery and ~60 min before the start of the experiment, 50-75 µCi was injected as a bolus dose (0.5 ml), followed by a continuous intravenous infusion of 51Cr-EDTA (10-30 µCi/ml in the Ringer solution) at a rate of 1.0 ml/h. Four 0.2-ml blood samples were drawn during the experiment at a time interval of ~30 min. The first one was taken 60 min after the injection of 51Cr-EDTA. After each blood sample withdrawal, the blood volume loss was compensated for by injection of a 10% Ficoll 400 solution in saline. The blood sample was centrifuged, and 50 µl of the plasma was removed for measurements of radioactivity [counts per minute (cpm)]. The gastric mucosa was covered with isotonic saline, which was replaced every 15 min. The luminal solution and the blood plasma were analyzed for 51Cr activity in a gamma counter (1282 Compugamma CS; Pharmacia, Uppsala, Sweden). In each experiment, the various blood plasma 51Cr-EDTA activities were plotted against time and a straight line was drawn between the two nearest values. Each clearance value was calculated by dividing each individual effluent cpm value by a corresponding plasma cpm value. If there was <10% deviation between the different blood plasma counts, a mean plasma cpm per milliliter value was calculated and used for all clearance samples. The part of the stomach that had been exposed in the chamber was cut out and weighed after the experiment. Clearance is expressed as milliliters per minute per 100 g wet tissue weight
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Blood Flow Measurements
LDF (Periflux Pf 2; Perimed, Stockholm, Sweden) was used for blood flow measurements in all experiments. The nature of the Doppler shift from an illuminated tissue depends on the velocity and number of moving red blood cells (23). The laser light (wavelength 633 nm, helium neon laser) is guided to the tissue by an optical fiber, and the backscattered light is picked up by a pair of fibers with a fiber separation of 0.7 mm. With this technique, blood flow is determined as a voltage output and expressed as percent of baseline values. Blood flow was recorded continuously from the mucosal side of the gastric mucosa, with the probe fixed to a micromanipulator (Leitz) and kept at a distance of 0.5-1 mm above the surface of the mucosa, in the saline solution. The accuracy of the LDF technique for the gastrointestinal application was evaluated and discussed earlier (1, 11, 20).Experimental Protocol
The animals were given at least 1 h to stabilize after the operation before the experiment was started. They were divided into seven groups, three for measurement of the epithelial cell surface pH (groups I-III) and four for assessment of blood flow and mucosal permeability (groups IV-VII).Groups I-III.
Before the experiments were started, systemic blood pressure and acid
output had been at steady state for 30 min. The H+
concentration at the epithelial cell surface was measured continuously, and values were noted every 5 min. In group I
(n = 6), the pH-sensitive electrode was inserted into
the mucus gel, with the electrode tip at the epithelial cell surface,
15 min after the start of the experiment. Thirty minutes after the
experiment was begun, 100 mM HCl was applied topically to the mucosa in
two successive 10-min periods. The topical HCl was then changed to
isotonic saline (after one rinse with saline), and the pH was followed
for another 30 min. In group II (n = 6),
pentagastrin was administered intravenously in a dose of 40 µg · kg1 · h
1 throughout
the experiment. Fifteen minutes after the start of pentagastrin
infusion, the pH-sensitive electrode was inserted into the mucus gel,
with the electrode at the epithelial cell surface. Thirty minutes after
the start of pentagastrin infusion, 100 mM HCl was applied topically to
the mucosa in two successive 10-min periods. The topical HCl was then
changed to isotonic saline (after one rinse with saline), and the pH
was followed for another 30 min. In group III
(n = 6), NaHCO3 (5 mmol · kg
1 · h
1) was
infused intravenously and continuously throughout the experiment. Fifteen minutes after the start of the HCO3
infusion,
the pH-sensitive electrode was inserted into the mucus gel, with the
electrode at the epithelial cell surface. Thirty minutes after the
start of the infusion of HCO3
, 100 mM HCl was applied
topically to the mucosa in two successive 10-min periods. The topical
HCl was then changed to isotonic saline (after one rinse with saline),
and pH was followed for another 30 min.
Groups IV-VII.
In all of these groups, after steady-state values of systemic blood
pressure, blood flow (LDF signal), and acid output had been recorded
for at least 30 min, 100 mM HCl was applied topically to the mucosa in
two successive 10-min periods followed by another 30-min control
period. Group IV (n = 6) had no further
treatment. In group V (n = 6), impromidine
was infused at 500 µg · kg1 · h
1 iv
throughout the experiment. Thirty minutes after the start of
impromidine infusion, 100 mM HCl was applied topically to the mucosa in
two successive 10-min periods followed by another 30-min control
period. In group VI (n = 6), pentagastrin
was administered in a dose of 40 µg · kg
1 · h
1 iv
throughout the experiment. Thirty minutes after the start of
pentagastrin infusion, 100 mM HCl was applied topically to the mucosa
in two successive 10-min periods followed by another 30-min control
period. In group VII, a continuous intravenous infusion of
NaHCO3 (5 mmol · kg
1 · h
1) was given
throughout the experiment (n = 6). Thirty minutes after
the start of the infusion of HCO3
, 100 mM HCl was
applied topically to the mucosa in two successive 10-min periods
followed by another 30-min control period.
Chemicals
Inactin was obtained from Research Biochemicals International (Natick, MA). Ficoll 400 was purchased from Sigma (St. Louis, MO). Heparin was obtained from KabiVitrum (Stockholm, Sweden). 51Cr-EDTA was purchased from NEN. The HCl-containing solution (100 mM) was made from a stock solution of 1 M HCl (Titrisol; Merck) and adjusted to isotonicity by the addition of NaCl. Tributylchlorosilane and H+ Ionophore II-Cocktail were obtained from Fluka (Buchs, Switzerland). HEPES was purchased from Sigma.Statistical Methods
The results are expressed as means ± SE. For statistical evaluations of differences, ANOVA for multiple comparisons followed by the Fisher's protected least significant differences test was performed. When abnormal distributions existed, the nonparametric Mann-Whitney test was used. When appropriate, Student's t-test for unpaired data was used. All statistical calculations were performed on a Macintosh with the software Statview II SE Graphics (Abacus Concepts, Berkeley, CA). The differences were regarded as significant if P < 0.05. ![]() |
RESULTS |
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Group I: Control Epithelial Cell Surface pH
Figure 1 shows the mean values for pH at the epithelial cell surface, as well as the acid secretion, in six untreated rats. Epithelial cell surface pH decreased significantly during application of 100 mM HCl compared with values before the acid period and compared with pentagastrin- and HCO3
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Group II: Pentagastrin Epithelial Cell Surface pH
Figure 2 presents the mean values for pH at the epithelial cell surface, as well as the acid secretion, in six pentagastrin-stimulated rats. During application of 100 mM HCl, there was a slight, but significant, decrease in the epithelial cell surface pH. Acid secretion was significantly higher in the pentagastrin-stimulated animals than in the control group (group I). Mucus gel thickness was 228 ± 35 µm during the control period. MAP was not significantly altered during the experiments (96 ± 4 mmHg during the control period, 95 ± 4 mmHg during topical administration of acid, and 97 ± 5 mmHg after acid exposure).
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Group III: NaHCO3 Epithelial Cell Surface pH
Figure 3 shows the mean values for pH at the epithelial cell surface, as well as the acid secretion, in six rats that received NaHCO3 intravenously. During application of 100 mM HCl, the epithelial cell surface pH was slightly but significantly reduced. However, after the acid was washed away the cell surface pH returned within 5 min to a level not significantly different from the period before acid exposure. Mucus gel thickness was 198 ± 58 µm during the control period. MAP was slightly but significantly increased during the experiments (93 ± 1 mmHg during the control period, 98 ± 1 mmHg during topical administration of acid, and 101 ± 1 mmHg after acid exposure).
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Group IV: Control LDF and Permeability
As seen in Fig. 4, the blood flow was increased to a maximum of +75% 5 min after application of acid in the six rats in which acid secretion was not stimulated. The blood flow was still significantly above the control level 20 min after the acid was changed to saline. The clearance from blood to lumen increased to a value three times above the control value of 0.2 ml · min
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Group V: Impromidine LDF and Permeability
The blood flow was increased to 35% above the control level 5 min after application of 100 mM HCl in the lumen in the impromidine-treated animals (Fig. 5). This blood flow increase was significantly smaller than that in the control experiments (group IV). The blood flow was not, however, altered by impromidine itself (94 ± 13% of control level before impromidine; not shown). The acid secretion was significantly higher in the impromidine-stimulated animals than in the control group (group IV). The clearance during the control period did not differ between this group and group IV, but the clearance was not altered during acid exposure as it was in group IV.
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Group VI: Pentagastrin LDF and Permeability
As seen in Fig. 6, the blood flow was increased ~25% above the control level 5 and 20 min after application of 100 mM HCl in the lumen in the pentagastrin-treated animals. The blood flow was not, however, altered by pentagastrin itself (114 ± 26% of the control level; not shown). Acid secretion was significantly higher in the pentagastrin-stimulated animals than in the control group (group IV). As in the other groups, the secretion was significantly increased after acid exposure, again most probably reflecting acid not totally washed away after the exposure. It is also seen in Fig. 6 that the clearance was not altered during acid exposure.
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Group VII: NaHCO3 LDF and Permeability
The blood flow increased by ~30% above the control level 10 min after application of 100 mM HCl in the lumen in the rats receiving intravenous NaHCO3 continuously (Fig. 7). This blood flow increase was significantly smaller than that found in the control experiments (group IV). The blood flow was not, however, altered by NaHCO3 in itself (102 ± 9% of control level before NaHCO3; not shown). Neither was the acid secretion altered by NaHCO3 (0.04 ± 0.03 µeq/min before and 0.01 ± 0.04 µeq/min during intravenous infusion of NaHCO3). The clearance during the control period did not differ between this group and group IV, but the clearance was not altered during acid exposure as it was in group IV (Fig. 7).
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DISCUSSION |
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In the present study, we have shown that in the acid-secreting
gastric mucosa the epithelial cell surface pH is neutral even when the
luminal pH is 1 (100 mM HCl). This is most probably due to transport of
HCO3 through the epithelial cells to buffer the
back-diffusing acid in the mucus at the epithelial cell surface. In the
nonstimulated rats with low acid secretion, the pH was significantly
reduced to ~1.6 ± 0.2 at the cell surface during application of
luminal HCl of pH 1 but returned toward the control level after the
exposure. Thus, in the absence of parietal cell production of
HCO3
, the surface epithelial cells were not able to
produce HCO3
in a sufficiently large amount to
maintain the previously described protective pH gradient in the mucus
(29, 30, 31, 36). When HCO3
was given
intravenously to rats with low spontaneous acid secretion, the surface
cell pH was only slightly reduced during application of acid (pH 1).
Thus the HCO3
infusion experiments confirm the
finding in the acid-secreting mucosae that blood-borne
HCO3
is important for transport into the mucus gel,
where it buffers the back-diffusing acid.
Moreover, during stimulation of acid secretion or during
HCO3 infusion and application of acid of pH 1 in our
rat model, no sign of disturbance of the permeability of the epithelial
cell lining was seen. However, when acid was applied in the
nonstimulated mucosa, H+ most probably entered the lamina
propria, thereby increasing the permeability of the cell lining. This
is in accordance with the observation by Kivilaakso et al.
(19) of a reduction in intramural pH during acid
application (120 mM) in nonstimulated rabbit mucosa but not in that
stimulated with histamine. Furthermore, O'Brien and Silen
(27) concluded from experiments in bullfrog fundic mucosa
that the secretory status of the mucosa is an important determinant of
the tolerance of the tissue to exogenous back-diffusion of
H+.
One explanation for the better resistance to luminal acid in an
acid-secreting than in a nonsecreting mucosa is that
HCO3 is transported across the basolateral membrane
of the parietal cells to the lamina propria simultaneously with acid
secretion into the gland lumen. The microvasculature of the gastric
mucosa is organized so that the HCO3
will be carried
from the parietal cells to the surface cells (5, 6). Thus,
during acid secretion, HCO3
not only alkalinizes the
lamina propria but is also available for transport across the surface
epithelial cells into the mucus gel and, as we have shown here,
increases the efficiency of the first line of defense. The epithelial
cell surface pH is slightly less reduced during luminal application of
acid in the acid-secreting stomachs (pentagastrin stimulated) than in
the HCO3
-treated rats. If the amount of
HCO3
added to the circulation, either by intravenous
infusion or by the parietal cells during acid secretion, is estimated,
it is obvious that the intravenous infusion might be 2-3 times
higher than the parietal cell production. However, the concentration or
delivery of HCO3
to the surface epithelial cells
might still be higher during acid secretion since the
HCO3
is produced in the epithelial cells and since
the systemic HCO3
is regulated and eliminated by the
kidneys. Furthermore, pentagastrin (gastrin) might also have other
protective properties, enhancing the possibility of maintaining a
neutral epithelial cell surface pH (32, 33).
Since we invariably found a neutral or slightly alkaline pH at the epithelial cell surface during acid secretion, the acid must have penetrated the mucus layer from the site of production to the lumen of the stomach without acidifying the epithelial cell surface. In earlier studies, we presented evidence for the existence of "channels" in the mucus for acid and pepsin transport from the gastric pit to the lumen of the stomach (10, 16). A low pH at the epithelial surface of acid-secreting stomachs was never observed in the present study, suggesting that acid is penetrating the mucus within structures not penetrated by our microelectrodes (tip diameter of 1-3 µm). The nature of these channels is at present being studied in our laboratory, and we have demonstrated slender and firm structures (16).
O'Brien and Bushell (26) reported that increasing the
HCO3 concentration on the serosal side in isolated
amphibian gastric mucosa reduced the back-diffusion of acid that had
been induced by passage of electrical current from the secretory to the
nutrient side in combination with HCl. The back-diffusion could not be prevented solely by increasing the pH to 8.2 on the nutrient side with
a buffer lacking HCO3
. This indicates the importance
of secreting the HCO3
through the surface epithelial
cells into the mucus, since this involves active transport processes
existing only for HCO3
. Furthermore, Kivilaakso
(18) found that intravenous infusion of NaHCO3
causing high-HCO3
metabolic alkalosis significantly
decreased the incidence of acid-induced mucosal injury. Interestingly,
low-HCO3
respiratory alkalosis of a similar degree
was not able to protect the mucosa against acid. These results indicate
that it is the presence of HCO3
rather than the
alkalization per se that protects the mucosa against acid.
Intraluminal application of acid with a pH of 1.7 has been shown to induce intracellular (surface cell) acidification both in the nonstimulated and in the pentagastrin-stimulated rat mucosa (24). The acidification was stronger in the nonstimulated mucosa, but distinct acidification was nevertheless observed in the pentagastrin-stimulated mucosa, suggesting that the pH gradient in the mucus might already have been destroyed at a pH of 1.7 in that rat model. Another study by the same group suggested that acid diffuses from its site of secretion toward the lumen, since they found an inverted pH gradient, with pH 5 in the lumen and pH 3.5 at the cell surface during pentagastrin stimulation (3). They have measured the gastric surface pH in rats using an inverted confocal microscope and a pH-sensitive dye (CI-NERF). In pilot experiments, we tried to mimic their experimental conditions with a high superfusion rate of Krebs saline (pH 5) in pentagastrin-stimulated rats (n = 2). During those conditions, we still measured a neutral pH (7.6 ± 0.1) at the epithelial cell surface. Hence, the superfusion of buffered luminal solutions at a high rate is not responsible for the differences in results between the study of Chu et al. and ours (3).
The gastric mucosal blood flow increased during acid instillation in
the corpus mucosa in the present study. This increase was significantly
attenuated during stimulation of acid secretion with pentagastrin or
impromidine (H2 receptor agonist) and during intravenous
infusion of NaHCO3 compared with the response to exposure to acid under resting conditions, again indicating a better resistance to acid in the acid-secreting stomach. Together, these results indicate
that acid will diffuse into the mucosa if it has a concentration high
enough to overcome the endogenous HCO3 secretion that
otherwise will neutralize the acid within the mucus gel. In our rat
preparation, 100 mM HCl would most likely enter the mucosa to signal
for an increase in blood flow. This blood flow increase was
significantly greater when the pH at the epithelial cell surface was
substantially reduced than when it was only slightly reduced.
A blood flow increase was also observed in the gastric mucosa of
urethan-anesthetized rats during application of 200 mM HCl (22) and was shown to be mediated by capsaicin-sensitive
afferent neurons. We have studies (17) showing that the
blood flow increase induced by acid in the lumen was not abolished by
indomethacin pretreatment, indicating that prostaglandins are not
involved in this hyperemic response. However, inhibition of the
endogenous production of nitric oxide (NO) by pretreatment with
N-nitro-L-arginine
prevented the blood flow increase induced by topical application
of acid. These findings are in agreement with results of a study by
Lippe et al. (21), who showed in anesthetized rats that
endothelium-derived NO plays an important role in gastric mucosal
vasodilation caused by back-diffusion of acid, whereas prostacyclins
are not involved. Holzer et al. (14) have demonstrated that back-diffused acid activates capsaicin-sensitive c-fibers. These,
in turn, release calcitonin gene-related peptide, a potent vasodilator
in the submucosa (7, 13). There is also strong evidence
for the involvement of NO in the hyperemic response to activation of
these c-fibers, because calcitonin gene-related peptide probably
releases NO (15, 37). Thus the hyperemia induced by
topically applied HCl in our studies most probably depends on NO
release, possibly through activation of capsaicin-sensitive c-fibers.
Disruption of the gastric mucosal barrier has been shown to lead to an
increase in gastric mucosal blood flow, which is thought to be a
defensive mechanism that minimizes further injury. Intraluminal acid in
combination with various agents such as (acetylsalicylic acid), ethanol, sodium taurocholate, or electric current have been used as barrier breakers (27, 28, 30). Guttu et al. (8) found that if 2 M NaCl was used as a barrier breaker
and the stomachs of anesthetized cats were perfused with HCl of pH 1 before and after application of the barrier breaker, the blood flow
increased approximately threefold after NaCl application and the
damaged mucosa was restored to a high degree. When the blood flow
increase was prevented, there was much less mucosal restitution, but
increasing the blood concentration of HCO3 completely
counteracted this effect. This is in conformity with our finding in the
present study that high blood concentrations of HCO3
,
achieved either by endogenous acid secretion or by intravenous infusion
of HCO3
, abolished the increase in
51Cr-EDTA clearance seen in the resting situation during
luminal application of acid. Furthermore, an increased blood flow was shown to enhance mucosal restitution and is hence beneficial to the
damaged mucosa (8). In the present study, the blood flow was increased to a significantly greater extent in the nonsecreting rats, in which the mucosal permeability had increased on intraluminal application of HCl.
In this study we have shown that stimulation of acid secretion protects
the corpus mucosa against luminal acid down to pH 1. The results
suggest that the defense mechanisms include HCO3
delivery by the blood from the parietal cells to the surface mucus
cells for transport into the mucus, where there is a preepithelial buffer layer. In the non-acid-secreting gastric mucosa, acid will most
probably diffuse from the lumen into the mucosa and thereby induce an
increase in the permeability of the mucosal lining, as well as a
substantial increase in blood flow.
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ACKNOWLEDGEMENTS |
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We thank Annika Jägare for excellent technical assistance.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (08646).
Address for reprint requests and other correspondence: L. Holm, Dept. of Physiology, Uppsala Univ., P.O.B. 572, S-751 23 Uppsala, Sweden (E-mail: Lena.Holm{at}Physiology.uu.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 October 1999; accepted in final form 16 August 2000.
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