PageArticles
1 of 35 in PresS. Am J Physiol Gastrointest Liver Physiol (May 18, 2006). doi:10.1152/ajpgi.00048.2006
Distinct Mechanisms of Acid-Induced HCO3- Secretion
in Normal and Slightly Permeable Stomachs
Eitaro Aihara, Yoko Sasaki, Fumitaka Ise, Kazutomo Kita
Yoko Nomura and Koji Takeuchi
Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical
University, Yamashina, Kyoto, Japan
Key words: gastric HCO3- secretion, slightly permeable stomach, mucosal acidification
capsaicin-sensitive afferent neurons, prostaglandin, nitric oxide, rat
Running title: HCO3- Secretion in Slightly Permeable Stomachs
Address correspondence to: Dr. Koji Takeuchi
Department of Pharmacology and Experimental Therapeutics
Kyoto Pharmaceutical University
Misasagi, Yamashina, Kyoto 607, Japan
Tel: (Japan) 075-595-4679; Fax: (Japan) 075-595-4774
E-mail: [email protected]
1
Copyright © 2006 by the American Physiological Society.
Page 2 of 35
Abstract
We investigated the regulatory mechanism of acid-induced HCO3- secretion in the slightly
permeable rat stomach following exposure to hyperosmolar NaCl.
Under urethane
anesthesia, a rat stomach was mounted on a chamber and perfused with saline, and the
secretion of HCO3- was measured at pH 7.0 using a pH-stat method and by adding 2 mM
HCl. Acidification of the normal stomach with 100 mM HCl increased HCO3- secretion,
and this response was totally inhibited by pretreatment with indomethacin but not
NG-nitro-L-arginine methyl ester (L-NAME) or chemical ablation of capsaicin-sensitive
afferent neurons. Exposure of the stomach to 0.5 M NaCl deranged the unstirred mucus
gel layer without damaging the surface epithelial cells. The stomach responded to 0.5 M
NaCl by secreting slightly more HCO3-, in an indomethacin-inhibitable manner, and
responded to even 10 mM HCl with a marked rise in HCO3- secretion, although 10 mM
HCl did not have an effect in the normal stomach. The acid-induced HCO3- response in
the NaCl-treated stomach was significantly but partially attenuated by indomethacin,
L-NAME or sensory deafferentation, and totally abolished when these treatments were
combined. These results suggest that gastric HCO3- secretion in response to acid is
regulated by two independent mechanisms, one mediated by prostaglandins (PGs) and
the other by sensory neurons and nitric oxide (NO). The acid-induced HCO3- secretion in
the normal stomach is totally mediated by endogenous PGs, but when the stomach is
made slightly permeable to acid, the response is markedly facilitated by sensory neurons
and NO.
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Introduction
The secretion of HCO3- plays a crucial role in protection of the gastroduodenal
mucosa from acid [5,10]. The secretion increases in response to mucosal acidification and
the process is mediated by multiple factors, including endogenous prostaglandins (PGs)
and nitric oxide (NO) as well as neuronal pathways [12,22,29,30]. Recently, we found that
the mechanism underlying acid-induced HCO3- secretion in the stomach differed
depending on the concentration of acid; the response caused by 100 mM HCl is mediated
only by PGs while that caused by 200 mM HCl is mediated by both capsaicin-sensitive
afferent neurons and NO, in addition to PGs [1]. It is assumed that 100 mM HCl applied
topically to the stomach increases PG biosynthesis by itself but does not acidify the mucosa
enough to activate the afferent neurons, resulting in an increase of HCO3- secretion, totally
dependent on endogenous PGs but not the afferent neurons or NO.
Mucus adherent to the luminal surface of the mucosa provides a zone of low
turbulence (unstirred layer), allowing the development of a gradient for HCO3- from the
luminal side [5,10,24]. Small amounts of HCO3- protect the mucosa against large amounts
of acid by neutralizing H+ ions that diffuse back into the mucus layer. Thus, it is possible
that the mucus gel layer hampers the penetration of luminal acid at 100 mM HCl, so that
the acid treatment at this concentration does not acidify the mucosa enough to activate
these afferent neurons. A hypertonic NaCl solution has been used to remove the mucus
gel layer from the surface epithelium [7]. In a preliminaly study, we observed that the
adherent mucus layer was removed following a brief exposure (10 min) of the stomach to
0.5 M NaCl, without accompanying any histological damage in the epithelial cells. This
technique provides conditions where the effect of luminal acid on the secretion of HCO3can be examined without disruption of the mucus layer.
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In the present study, we examined the effect of mucosal acidification on HCO3secretion in the stomach following a brief exposure to 0.5 M NaCl and investigated the
regulatory mechanism of this response in the slightly permeable stomach, in comparison
with the intact stomach.
Materials and Methods
Animals
Male Sprague Dawley rats (220-260 g, Nippon Charles River, Shizuoka, Japan)
were used. The animals were deprived of food but allowed free access to tap water for 18
hr before the experiments. All studies were performed under urethane anesthesia (1.25
g/kg, i.p.) using 4~8 animals per group. All experimental procedures described here were
approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical
University.
Determination of Gastric HCO3- Secretion
Gastric HCO3- secretion was measured in the chambered stomach as described
previously [22,28]. In brief, the abdomen was incised and the stomach was exposed,
mounted on a chamber (the exposed area; 3.1 cm2), and superfused with isotonic saline
(154 mM NaCl) that was gassed with 100% O2 and kept in a reservoir. The secretion of
HCO3- was measured at pH 7.0 by using a pH-stat method (Hiranuma Comtite-8, Mito,
Japan) and by adding 2 mM HCl to the reservoir. To unmask HCO3- in the stomach, the
acid secretion was completely inhibited by omeprazole given i.p. at a dose of 60 mg/kg.
Omeprazole at this dose has been shown to have no influence on gastric HCO3- secretion in
rats [6]. After basal HCO3- secretion had well stabilized, the mucosa was exposed to
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10~200 mM HCl for 10 min, and the secretion of HCO3- was measured before and after the
exposure. After the application of HCl, the mucosa was rinsed several times with saline,
another 2 ml of saline was instilled and the perfusion was resumed. The rate of HCO3secretion was low for a little while after the acidification but gradually increased, reaching
a plateau levels within 5~10 min thereafter. In half the number of animals subjected to
the acid treatment, the stomach was exposed to 0.5 M NaCl for 10 min, immediately before
the acidification (5~100 mM HCl). In some cases, the effect of NOR-3 (a NO donor) on
HCO3- secretion was examined. This agent (3 mg/ml) was applied topically to the chamber
for 10 min. In addition, the effects of several treatments on the acid-induced change in
HCO3- secretion were examined in the stomach with or without prior exposure to 0.5 M
NaCl. Indomethacin (5 mg/kg) was given s.c. 30 min before the acidification while
NG-nitro L-arginine methyl ester (L-NAME: 20 mg/kg) was given s.c. 3 hr before, because
this agent acutely increased HCO3- secretion through a neural reflex due to an increase of
blood pressure [2,26].
Chemical ablation of capsaicin-sensitive afferent neurons was
achieved with repeated s.c. injections of capsaicin (total dose; 100 mg/kg) once daily for 3
days 2 weeks before the experiment [15,22,30]. The injections were performed under
ether anesthesia, and the rats were pretreated with terbutaline (0.1 mg/kg, i.m.) and
aminophylline (10 mg/kg, i.m.) to counteract the respiratory impairment associated with
capsaicin. To check for the effectiveness of the treatment, a drop of capsaicin solution (0.1
mg/ml) was instilled onto one eye of each rat, and wiping movements were counted as
previously reported.
In another experiments, we measured the amount of luminal acid loss when the
mucosa was exposed to 50, 100 and 200 mM HCl without or 50 mM HCl with prior
exposure to 0.5 M NaCl, in the animals pretreated with omeprazole to inhibit acid
secretion. The stomach was exposed to 2 ml of 50-200 mM HCl solution for 10 min, then
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the solution was recovered. Luminal acid loss was determined from analysis of the
collected acid solution. Each sample was analyzed for volume and acid concentration,
which was determined by automatic titration of an aliquot with 50 mM NaOH to pH 7.0
(Autoburette, Comtite-7, Hiranuma, Tokyo, Japan). The amount of luminal acid loss was
calculated as the difference between the product of the final volume and concentration and
the product of the initial volume and concentration. In half the number of animals, the
stomach was exposed for 10 min to 2 ml of 0.5 M NaCl, immediately before the acid
treatment.
Determination of Gastric Potential Difference (PD)
The methods used for determining transmucosal PD and gastric perfusion were
as described in our previous paper [32]. The animals were pretreated with omeprazole
(60 mg/kg, i.p.) to inhibit acid secretion. The abdomen was opened through a midline
incision, and the stomach exposed, mounted on an ex-vivo chamber, and superfused at a
flow rate of 1 ml/min with isotonic saline kept in a reservoir. The PD was determined
using two agar bridges, one positioned in the chamber and the other in the abdominal
cavity. Changes in PD were monitored continuously on a two-pen recorder (U-228,
Tokai-irika, Tokyo, Japan). After the basal PD had stabilized, the perfusion system was
interrupted, and the solution in the chamber was withdrawn. The mucosa was then
exposed for 10 min to 2 ml of 0.5 M NaCl. After the application of NaCl, the mucosa was
rinsed with saline, another 2 ml of saline was instilled and the perfusion was resumed.
Histological Evaluation of Gastric Mucosa
The gastric mucosa was examined with a light microscope after exposure to 0.5 M NaCl
for 10 min. The mucosa was excised immediately after the NaCl treatment, and the tissue
sample was immersed in 10% formalin, sectioned at 5 μm, and stained with hematoxylin &
eosin as well as PAS.
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Determination of Mucosal PGE2 Content
Mucosal PGE2 levels were determined in the stomach under various conditions.
The stomach mounted on a chamber was exposed for 10 min to 0.5 M NaCl, 10 mM HCl or
100 mM HCl. In some cases, the mucosa was exposed to 10 mM HCl immediately after
the NaCl treatment. Indomethacin (5 mg/kg) was given s.c. 1 hr before NaCl or HCl
treatment. Immediately after the exposure, the corpus mucosa was excised, weighed,
and put in a tube containing 100% methanol plus 0.1 M indomethacin [9]. Then, the
samples were minced with scissors, homogenized, and centrifuged for 10 min at 12000
r.p.m. at 40ºC. The supernatant of each sample was used for the determination of PGE2
by EIA using a PGE2-kit (Cayman Chemical Co., Ann Arbor, MI).
Measurement of Luminal NO Content
The release of NO from the stomach was determined indirectly as an amount of
NO metabolites, NO2- and NO3-. The stomach mounted on a chamber was exposed for 10
min to 0.5 M NaCl or 10~200 mM HCl, and perfused with saline before and after the
exposure. In some cases, the mucosa was exposed to 10 mM HCl immediately after the
NaCl treatment. The perfusate collected for 30 min before and after the exposure was
centrifuged for 15 min at 3000 r.p.m. and stored at –80ºC until the assay. NO was
measured in aliquots of the samples by the Griess method (11) after reduction of nitrate to
nitrite with nitrate reductase. Nitrites were incubated with Griess reagent (0.1%
naphthylene diamine dihydrochloride and 1% sulfanilamide in 2.5% H3PO4) for 10 min at
room temperature, and the absorbance was measured at 550 nm. L-NAME (20 mg/kg)
was given s.c. 3 hr before the exposure to 0.5 M NaCl plus 10 mM HCl.
Preparation of Drugs
The drugs used were urethane (Tokyo kasei, Tokyo, Japan), hydrochloric acid
(HCl) and sodium chloride (NaCl)(Nacalai tesque, Kyoto Japan), NG-nitro L-arginine
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methyl ester (L-NAME) and indomethacin (Sigma Chemicals, St. Louis, MO), and NOR-3
[(±)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine](Dojindo, Kumamoto, Japan).
Capsaicin was dissolved in a Tween 80-ethanol solution (10% ethanol, 10% Tween and
80% saline, w/w: Wako, Osaka, Japan). NOR-3 was first dissolved in dimethyl sulfoxide
(DMSO) and then diluted with saline to the desired concentration. Other agents were
dissolved in saline. Each agent was prepared immediately before use and given in a
volume of 0.5 ml per 100 g body weight in the case of i.p. or s.c. administration, or in a
volume of 0.1 ml per 100 g body weight in the case of i.m. administration, or applied
topically to the chamber in a volume of 2 ml per rat. Control animals received saline in
place of active agents.
Statistics
Data are presented as the mean±SE from 4~8 animals per group. Statistical
analyses were performed using a two-tailed Dunnett’s multiple comparison test, and
values of p<0.05 were regarded as significant.
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Results
Effect of Mucosal Acidification on HCO3- Secretion in Normal Stomachs
Under urethane anesthesia, the rat stomach spontaneously secreted HCO3- at a
rate of 0.2~0.4 μEq/10 min during the test period. Mucosal exposure to acid (10~100 mM
HCl) for 10 min increased the secretion of HCO3- in a concentration-dependent manner,
the net HCO3- output at 200 mM HCl being 1.8±0.2 μEq/hr (Figure 1). The HCO3response to 100 mM HCl was all but totally inhibited by prior administration of
indomethacin (5 mg/kg, s.c.) but not significantly affected by either L-NAME (20 mg/kg,
s.c.) or chemical ablation of capsaicin-sensitive afferent neurons (Figure 2A). By contrast,
the response induced by 200 mM HCl was significantly mitigated by both indomethacin
and L-NAME as well as sensory deafferentation (Figure 2B). When the inhibitory effect of
indomethacin was compared for the responses to 100 mM and 200 mM HCl, it was evident
that the effect was much greater in the case of the response to 100 mM HCl.
Effect of Hypertonic NaCl on HCO3- Secretion in Stomachs
Under chambered conditions in the presence of omeprazole (inhibition of acid
secretion), the rat stomach generated a PD of -50~55 mV (mucosa negative) and
maintained relatively constant values during the test period. Mucosal exposure to 0.5 M
NaCl for 10 min caused a slight but significant reduction in the transmucosal PD from
52.6±3.1 to 44.3±2.6 mV, immediately after the exposure, and the reduced PD quickly
reverted after the removal of NaCl from the chamber, the values reaching approximately
80% of the pre-exposure level within 60 min. The mucosal exposure to 0.5 M NaCl for 10
min caused no damage in the mucosa visible macroscopically 1 hr after the exposure.
Histologically, the surface epithelial cells maintained a continuity even after exposure to
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0.5 M NaCl, and the amount of the PAS-positive substance adherent to the surface cells
was slightly reduced (Figure 3). The mucosal permeability to hydrogen ion was also
examined in the normal and 0.5 M NaCl-treated stomach, in the absence of endogenous
acid secretion. When the mucosa was exposed to 50, 100 and 200 mM HCl for 10 min in
normal stomachs, the amount of luminal acid loss was 7.5±2.6, 15.3±3.7 or 36.4±5.8 μEq/10
min, respectively. Luminal acid loss observed during exposure to 50 mM HCl was
markedly increased in the stomach pre-exposed to 0.5 M NaCl, reaching the values of
23.6±1.9 μEq/10 min, approximately 3 or 1.5 times greater than those observed at 50 mM
or 100 mM HCl in the normal stomach, respectively. Although the acid loss observed in
the 0.5 M NaCl-treated stomach during exposure to 50 mM HCl was little lower than that
observed in the normal stomach exposed to 200 mM HCl, there was no statistically
significant difference between these two groups.
Following exposure to 0.5 M NaCl for 10 min, the rate of HCO3- secretion was
slightly but significantly increased to 170% of basal levels, the net HCO3- output being
0.6±0.1 μEq/hr (Figure 4A).
This response was almost totally prevented by prior
administration of indomethacin (5 mg/kg, s.c.), the inhibition being over 90% (Figure 4B).
By contrast, the response to 0.5 M NaCl was not significantly affected by either prior
administration of L-NAME or chemical ablation of capsaicin-sensitive afferent neurons.
Mucosal Acidification on HCO3- Secretion in Stomachs Pretreated with
Hypertonic NaCl
The secretion of HCO3- in the stomach exposed for 10 min to 0.5 M NaCl was
further enhanced by subsequent acidification of the mucosa (5~50 mM HCl) in a
concentration-dependent manner; the net HCO3- output induced by 50 mM HCl was
equivalent to that caused by 200 mM HCl in normal stomachs (Figure 5).
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The secretion
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of HCO3- was not further enhanced by acidification of the mucosa with 100 mM HCl, the
net HCO3- output being equivalent to that obtained with 50 mM HCl (data not shown).
When the net HCO3- output caused by acidification in normal and 0.5 M NaCl-treated
stomachs was plotted against various concentrations of HCl, it was found that the curve
obtained in the latter stomach shifted to the left., ie., lower concentrations of HCl (Figure
6).
The increased HCO3- secretion caused by 0.5 M NaCl was further enhanced by
subsequent exposure to 10 mM HCl for 10 min, the net HCO3- output being 1.4±0.2 μEq/hr,
and this response was also totally inhibited by pretreatment with indomethacin (Figure 7).
Likewise, the response to 10 mM HCl was significantly but partially attenuated by both
L-NAME (20 mg/kg, s.c.) and sensory deafferentation, the degree of inhibition by these
two treatments being equivalent, about 60%.
Mucosal PGE2 Content in the Stomach Following Acidification with or
without Hypertonic NaCl Treatment
The level of PGE2 in the gastric mucosa of normal rats was 7.2±0.6 ng/g tissue.
Mucosal exposure to 0.5 M NaCl for 10 min stimulated the generation of PGs to
significantly increase the PGE2 content of the stomach (Figure 8). In contrast, exposure to
10 mM HCl for 10 min did not affect PG production in the stomach, and the mucosal PGE2
content remained at the level in control stomachs. However, the PGE2 content was
significantly increased after exposure of the mucosa to 100 mM HCl, the value being about
2 times the control level, and the effect was equivalent to that of 0.5 M NaCl. Prior
administration of indomethacin significantly inhibited the increase in mucosal PGE2
content following exposure to 0.5 M NaCl or 100 mM HCl. The combined exposure to 0.5
M NaCl and 10 mM HCl also significantly increased mucosal PGE2 content, yet the degree
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of the increase was almost equivalent to that induced by 0.5 M NaCl alone.
Luminal Release of NO in the Stomach Following Acidification with or
without Hypertonic NaCl Treatment
The amount of NO2-/NO3- in the gastric lumen of saline-treated rats was 14.0±0.4
nmol/30 min. The luminal NO level remained unchanged following exposure of the
mucosa to 0.5 M NaCl or 10-100 mM HCl for 10 min, but significantly increased after the
exposure to 200 mM HCl; the value was 17.2±0.2 nmol/30 min, about 42.1% increase of the
control value (Figure 9). In the stomach pretreated with 0.5 M NaCl, however, the
luminal release of NO was significantly increased after exposure to 10 mM HCl for 10 min;
the values were 17.2±0.2 nmol/30 min, almost equivalent to those observed after exposure
to 200 mM HCl alone. This response was totally attenuated by prior administration of
L-NAME (20 mg/kg, s.c.).
Effect of NOR-3 on Gastric HCO3- Secretion and PGE2 levels
Since the HCO3- response induced by 10 mM HCl in the 0.5 M NaCl pre-exposed
stomach was significantly inhibited by L-NAME, it is possible that NO also stimulates the
secretion of HCO3- in the stomach, as in the duodenum [22]. To confirm the stimulatory
role of NO in gastric HCO3- secretion, the effect of the NO donor NOR-3 on the secretion
was examined. NOR-3 applied to the chamber for 10 min increased the secretion of
HCO3- in the stomach from 0.2±0.1 μEq/10 min to 0.5±0.1 μEq/10 min, the net HCO3output being 1.0±0.2 μEq/hr.
This response was significantly inhibited by prior
administration of indomethacin, the net HCO3- output being 0.3±0.1 μEq/hr. This agent
also increased PGE2 production in the gastric mucosa, and the PGE2 content was 7.4±0.5
ng/g tissue, which is significantly greater than that (4.9±0.4 ng/g tissue) in the control
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stomach.
The stimulatory effect of NOR-3 on PGE2 production was almost totally
attenuated by prior administration of indomethacin.
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Discussion
The secretion of HCO3- from the gastroduodenal mucosa increases in response to
luminal acid and this response plays a particularly important role in protecting the mucosa
from acid [25]. Previous studies demonstrated that this process in the duodenum is
regulated by multiple factors, including PGs, NO and sensory neurons [12,22,29,30], yet
there is limited information on the response in the stomach.
HCO3- secretion in the duodenum was increased by acidification of the mucosa
even with 10 mM HCl, and this response was blocked by both indomethacin, L-NAME
and the ablation of capsaicin-sensitive afferent neurons [22,26]. In the stomach, however,
the response occurred on acidification with 50 mM or more of HCl, suggesting a different
threshold for the response as compared to the duodenum [1,26]. Furthermore, we have
recently found that the mechanism underlying the response in the stomach differed
depending upon the degree of acidification., ie, the concentration of the acid solution to
which the mucosa was exposed [1]. The response caused by 100 mM HCl is mediated only
by PGs while that caused by 200 mM HCl is mediated by both capsaicin-sensitive afferent
neurons and NO, in addition to endogenous PGs. We previously examined the effect of
HCl at various concentrations (0.1~0.35 M) on transmucosal PD of rat stomachs and found
that the PD was not decreased by HCl even at 0.25 M [27]. Thus, it is likely that 200 mM
HCl, unlike 0.5 M NaCl, does not damage the surface epithelial cells.
Indeed, we
reported that gastric HCO3- secretion in response to 200 mM HCl was completely
attenuated by sacrifice of the animal by saturated KCl [1], suggesting a totally active
process of this secretion. On the other hand, capsaicin-sensitive afferent neurons are
activated not only by capsaicin but also by hydrogen ions as well, resulting in the
liberation of calcitonin gene-related peptide (CGRP) from the nerve ending, which then
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stimulates the production/release of NO [19]. These results led us to speculate that 100
mM HCl applied topically to the stomach increases PG biosynthesis but does not acidify
the mucosa enough to activate the afferent neurons, resulting in an increase of HCO3secretion totally mediated by endogenous PGs but not NO or the afferent neurons. By the
way, the secretion of mucus is also stimulated by both PGs, NO and sensory neurons,
similar to the secretion of HCO3- [3,16,23]. Therefore, it is possible that below a
concentration of 100 mM, the acid is captured, in large part, by the mucus-HCO3- barrier
and does not acidify the mucosa enough to stimulate these afferent neurons.
In the present study, we found that a brief exposure (10 min) of the mucosa to 0.5
M NaCl increased the basal rate of HCO3- secretion in the stomach.
Although the
application of the NaCl solution, which is slightly hypertonic (3.2 times), caused a small
reduction in the gastric PD, this treatment did not damage the surface epithelium
histologically and only deranged the mucus gel layer. In the present study, gastric PD
was measured in the animals pretreated with omeprazole to inhibit acid secretion. Thus,
the changes in PD observed after 0.5 M NaCl treatment would reflect mostly those in
tissue electrical resistance as well as those in electrode/solution junction potentials.
Because mucosal exposure to 0.5 M NaCl apparently sloughed off the mucus layer, it may
be assumed that a small decrease in PD after 0.5 M NaCl treatment is caused by changes in
liquid junctional potentials due to derangement of mucus layer. On the other hand, it is
known that hypertonic saline acts as a mild irritant to the stomach and induces adaptive
cytoprotection mediated by endogenous PGs [20,21]. As expected, we found that the
mucosal exposure to 0.5 M NaCl significantly increased the PGE2 content of the stomach.
In addition, the secretion of HCO3- in response to 0.5 M NaCl was totally prevented by
prior administration of indomethacin, suggesting that HCO3- appeared in the lumen via an
active process mediated by endogenous PGs and not through passive diffusion. We
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previously reported that gastric alkaline response occurred in the stomach after exposure
to 1 M NaCl or 20 mM taurocholate was not affected by indomethacin, suggesting a
passive diffusion of HCO3- [20,32,33]. Certainly, after exposure of the stomach to these
irritants the PD was markedly reduced and the surface epithelial cells were extensively
sloughed. Since the response to 0.5 M NaCl was not affected by either L-NAME or the
ablation of capsaicin-sensitive afferent neurons, it is assumed that neither NO nor sensory
neurons are involved in the regulatory mechanism of this process.
Of the most important findings of the present study is that the threshold
concentration of acid required for stimulating HCO3- secretion was significantly decreased
in the permeable stomach following exposure to 0.5 M NaCl. The minimum concentration
of acid required for stimulating HCO3- secretion in the stomach pre-exposed to 0.5 M NaCl
was 5 mM, roughly 10 times less than that required in the normal stomach. Furthermore,
the HCO3- response induced by 10 mM HCl was significantly abrogated by prior
administration of L-NAME and sensory deafferentation, suggesting the involvement of
NO and sensory neurons in this process. The mucosal PGE2 content significantly increased
after treatment with 0.5 M NaCl and was not further enhanced by additional exposure to
10 mM HCl. It is possible that 0.5 M NaCl treatment enhances the epithelial permeability
to increase acid back-diffusion, resulting in acidification of the mucosa. As expected, acid
loss from the lumen during exposure to 50 mM HCl was markedly increased in the
stomach pre-exposed to 0.5 M NaCl, reaching the values of 23.6±1.9 μEq/10 min,
approximately 3 or 1.5 times greater than those observed in the normal stomach exposed
to 50 mM or 100 mM HCl, respectively, and little lower than those observed during
exposure to 200 mM HCl. Thus, a threshold concentration of HCl for acidification of the
mucosa efficiently to stimulate the HCO3- secretion mediated by both PG and sensory
neurons is between 100 mM and 200 mM HCl in the normal stomach. In addition, since
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0.5 M NaCl treatment sloughed off the mucus layer without damaging the surface cells, it
is assumed that luminal acid can acidify the mucosa efficiently without being trapped with
the mucus gel. Because of these two factors, even a low concentration of acid can acidify
the mucosa sufficiently to activate afferent neurons. It is known that endogenous PGs
play a crucial role in the gastric functional responses induced by these afferent neurons,
probably sensitizing the neurons through EP1 receptors [17,34]. In addition, activation of
these afferent neurons causes CGRP, which in turn stimulates the production and release
of NO [26]. Indeed, we observed that the luminal release of NO remained unchanged
after exposure of the stomach to 0.5 M NaCl or 10-100 mM HCl alone, yet significantly
increased after exposure to 10 mM HCl in the 0.5 M NaCl-treated stomach. Thus, it is
understandable that indomethacin totally inhibited the secretion of HCO3- in the
permeable stomach while both L-NAME and sensory deafferentation only partial
attenuated the response.
At present, how acidification stimulates NO release in the stomach or which cell
type is responsible for NO release remains unknown. Various types of cells including
surface epithelial cells, enteric neurons and endothelial cells are capable of producing NO.
Brown et al [4] reported that cells in the elutriator fraction rich in mucous-epithelial cells
exhibit the most activity of the constitutive type of NO synthase in the stomach, localizing
in parallel with the NADPH-dependent diaphorase activity.
On the other hand,
acid-induced HCO3- secretion is mediated via an axonal reflex pathway, in addition to
endogenous PGs and NO [13,22,29,30]. In the present study, this process was indeed
attenuated by the functional ablation of capsaicin-sensitive sensory neurons.
Other
studies also showed that the gastric hyperemic response induced by acid back-diffusion is
mediated by NO released by the stimulation of sensory neurons [14,18]. Thus, it is also
possible that mucosal acidification increases NO release through stimulation of
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capsaicin-sensitive sensory neurons. On the other hand, several studies reported that NO
or NO donors stimulate PG production in various organs and cells [8,35,36]. Uno et al.
[35] reported that the NO donor, S-nitroso-N-acetyl-penicillamine, stimulates PGE2
production in rat gastric epithelial cells. Furukawa et al. [8] reported that both NOR-3 and
dbcGMP increased PGE2 production in isolated bullfrog duodenums, suggesting a
NO/cGMP-dependent increase in PG production. In the present study, the increased PGE2
generation after the mucosal acidification was blocked not only by indomethacin but also
by L-NAME. In addition, we also observed that NOR-3, at a dose that stimulated HCO3secretion, increased PGE2 production in the gastric mucosa, and these effects were both
suppressed by indomethacin. These results strongly suggest an interactive role for NO
and PGs in the acid-induced secretion of HCO3- in the stomach.
Given the above findings, the present study suggests that gastric HCO3- secretion
in response to acid is regulated by two independent mechanism; one mediated by PGs and
the other by sensory neurons and NO. Normally, acid-induced HCO3- secretion is
mediated entirely by endogenous PGs, but when the stomach is made slightly permeable
to acid, as induced by exposure to irritating substances, the response is markedly
facilitated by the activation of sensory neurons and NO, suggesting the co-operative action
of these three factors.
Acknowledgements
This research was supported in part by the Kyoto Pharmaceutical University’s
“21st Century COE” program and the “Open Research” Program from the Ministry of
Education, Science and Culture of Japan.
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Figure Legends
Figure 1.
Effect of mucosal acidification on HCO3- secretion in normal rat stomachs.
Mucosal acidification was achieved through exposure of the stomach to various
concentrations of acid (10~200 mM HCl) for 10 min, and the secretion of HCO3- was
measured before and after the exposure. A: Data indicate the percentage of basal HCO3secretion and are presented as the mean±SE of values determined every 10 min from 4~6
rats. B: Data show total net HCO3- output for 1 hr after treatment with saline or acid and
are presented as the mean±SE from 4~6 rats. *Significant difference from control, at
p<0.05.
Figure 2.
Effect of indomethacin, L-NAME or chemical ablation of sensory neurons on
the secretion of HCO3- induced by acidification with 100 mM HCl (A) and 200 mM HCl (B)
in normal rat stomachs. The secretion was stimulated by exposure of the stomach to 100
mM or 200 mM HCl for 10 min, and was measured before and after the acidification.
Indomethacin (5 mg/kg) or L-NAME (20 mg/kg) was given s.c. 30 min or 3 hr,
respectively, before the acidification. Chemical ablation of sensory neurons was achieved
with 3 consecutive s.c. injections of capsaicin (total 100 mg/kg) 2 weeks before the
experiment (capsaicin pretreatment). Data show total net HCO3- output for 1 hr after
treatment with acid and are presented as the mean±SE from 4~6 rats.
Significant
difference at p<0.05; * from control; # from vehicle.
Figure 3. Histology of the gastric mucosa after exposure to 0.5 M NaCl for 10 min (HE
and PAS stainings). The mucosa was excised immediately after exposure to 0.5 M NaCl.
23
Page 24 of 35
Figures show; A: normal mucosa (H&E), B: 0.5 M NaCl-treated mucosa (H&E), C: normal
mucosa (PAS), D: 0.5 M NaCl-treated mucosa (PAS).
Figure 4.
A: Effect of mucosal application of 0.5 M NaCl on HCO3- secretion in rat
stomachs. The stomach was exposed to 0.5 M NaCl for 10 min, and HCO3- secretion was
measured before and after the exposure. Data are presented as % basal HCO3- secretion
and represent the mean±SE of values determined every 10 min from 4~6 rats.
*
Significant difference from control, at P<0.05. B: Effect of indomethacin, L-NAME or
chemical ablation of sensory neurons on the response induced by mucosal exposure to 0.5
M NaCl in rat stomachs. Indomethacin (5 mg/kg) or L-NAME (20 mg/kg) was given s.c.
30 min or 3 hr, respectively, before exposure to 0.5 M NaCl. Chemical ablation of sensory
neurons was achieved with 3 consecutive s.c. injections of capsaicin (total 100 mg/kg) 2
weeks before the experiment (capsaicin pretreatment). Data show total net HCO3- output
for 1 hr after exposure to 0.5 M NaCl and are presented as the mean±SE from 4~6 rats.
Significant difference at p<0.05; * from control; # from vehicle.
Figure 5.
Effect of mucosal acidification on HCO3- secretion in rat stomachs pretreated
with 0.5 M NaCl. The stomach was exposed to 0.5 M NaCl for 10 min, immediately
before acidification of the mucosa with various concentrations of acid (5~50 mM HCl), and
HCO3- secretion was measured before and after the treatment. A: Data are presented
as % basal HCO3- secretion and represent the mean±SE of values determined every 10 min
from 4~6 rats. B: Data show total net HCO3- output for 1 hr after the acidification and are
presented as the mean±SE from 4~6 rats. Significant difference at p<0.05; * from control;
# from saline.
24
Page 25 of 35
Figure 6. The secretion of HCO3- in response to various concentrations of HCl in normal
and 0.5 M NaCl-treated stomachs. Values are taken from those in Figures 1 and 5, and
represent the net HCO3- output for 1 hr induced by exposure for 10 min to various
concentrations of HCl (10~100 mM).
Note that the concentration-response curve is
shifted to the left in the stomach treated with 0.5 M NaCl. Data are presented as the mean
from 4~6 rats. * Significant difference from values in normal stomach, at P<0.05.
Figure 7.
Effect of indomethacin, L-NAME or chemical ablation of sensory neurons on
HCO3- secretion induced by acidification with 10 mM HCl in the rat stomach pretreated
with 0.5 M NaCl. The stomach was exposed to 0.5 M NaCl for 10 min, immediately
before acidification of the mucosa with 10 mM HCl, and HCO3- secretion was measured
before and after the treatment. Indomethacin (5 mg/kg) or L-NAME (20 mg/kg) was
given s.c. 30 min or 3 hr, respectively, before the acidification. Chemical ablation of
sensory neurons was achieved with 3 consecutive s.c. injections of capsaicin (total 100
mg/kg) 2 weeks before the experiment (capsaicin pretreatment). Data show total net
HCO3- output for 1 hr after treatment with acid and are presented as the mean±SE from
4~6 rats. Significant difference at p<0.05; * from saline; # from vehicle.
Figure 8. Changes in mucosal PGE2 levels after acidification of the rat stomach, with or
without prior exposure to 0.5 M NaCl. The mucosa was exposed for 10 min to 0.5 M
NaCl, 10 mM HCl or 100 mM HCl. In some cases, the mucosa was first exposed for 10
min to 0.5 M NaCl followed immediately by the 10 min-exposure to 10 mM HCl.
Indomethacin (5 mg/kg) was given s.c. 30 min before the acidification. The mucosa was
excised immediately after the above treatment, and PGE2 levels were determined by EIA.
Data are presented as the mean±SE from 5~6 rats. Significant difference at p<0.05; * from
25
Page 26 of 35
control; # from vehicle.
Figure 9. Changes in luminal NO release after acidification of the rat stomach, with or
without prior exposure to 0.5 M NaCl (A) and the effect of L-NAME on the response
induced by 0.5 M NaCl plus 10 mM HCl. The mucosa was exposed for 10 min to 0.5 M
NaCl, 10 mM HCl or 100 mM HCl. In some cases, the mucosa was first exposed for 10
min to 0.5 M NaCl followed immediately by the 10 min-exposure to 10 mM HCl.
L-NAME (20 mg/kg) was given s.c. 3 hr before the acidification. The perfusate was
collected for 30 min before and after the acidification, and NO was measured by the Griess
method. In Fig. A, the data show the values obtained after the exposure and are presented
as the mean±SE from 4~5 rats. *Significant difference from normal at P<0.05. In Fig. B,
the data show the values obtained before and after the exposure, and represent the
mean±SE from 4~5 rats. Significant difference at P<0.05; *from normal; #from Before.
26
Page 27 of 35
Figure 1
27
-
0.5
#
*
1.0
*
0
100 mM HCl
-0.5
-0.5
Figure 2
28
1.5
1.0
0.5
*#
Capsaicin
Pretreatment
*
L-NAME
B
Indomethacin
2.0
Vehicle
N=4~6
# * P<0.05
Control
-
*
HCO3 Output (μEq/hr)
Cap. pretreat.
L-NAME
Indomethacin
A 1.5
Vehicle
Control
HCO3 Output (μEq/hr)
Page 28 of 35
N=4~6
#
* P<0.05
*#
*#
0
200 mM HCl
Page 29 of 35
Figure 3
29
Page 30 of 35
Figure 4
30
Page 31 of 35
Figure 5
31
Page 32 of 35
Normal
0.5 M Treated
1.0
*
N=4~6
P<0.05
*
*
*
-
Net HCO3 Output (μEq/hr)
1.5
0.5
0
0
5
10
50
100
Concentration of HCl (mM)
Figure 6
32
Page 33 of 35
Figure 7
33
Page 34 of 35
Figure 8
34
Page 35 of 35
140
120
*
100
60
40
20
0
10
100
200
0.5 M NaCl
HCl (mM)
NOx output (nmol/ml/hr)
B
120
10 mM HCl
80
Normal
NOx output (nmol/ml/hr)
*
N=4~5
* P<005
Vehicle
A
#
Before
After
*
100
N=4~5
#
* P<005
80
60
*
40
*
20
0
Normal
Control
L-NAME
0.5 M NaCl+10 mM HCl
Figure 9
35