Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study was undertaken to determine the mechanism by which ammonium chloride (NH4Cl) inhibits stimulated acid secretion in the bullfrog gastric mucosa. To this end, four possible pathways of inhibition were studied: 1) blockade of basolateral K+ channel, 2) blockade of ion transport activity, 3) neutralization of secreted H+ in the luminal solution, or 4) ATP depletion. Addition of nutrient 10 mM NH4Cl (calculated NH3 concentration = 92.5 µM and NH4+ concentration = 9.91 mM) inhibited acid secretion within 30 min. Inhibition of acid secretion did not occur by blockade of basolateral K+ channel activity or ion transport activity or by neutralization of the luminal solution. Although ATP depletion occurred in the presence of NH4Cl, the magnitude of ATP depletion in 30 min was not sufficient to inhibit stimulated acid secretion. By comparing the effect of NH4Cl on the resistance of inhibited or stimulated tissues, we demonstrate that NH4Cl acts specifically on stimulated tissues. We propose that NH4Cl blocks activity of an apical K+ channel present in stimulated oxyntic cells. Our data suggest that the activity of this channel is important for the regulation of acid secretion in bullfrog oxyntic cells.
Rana catesbeiana; gastric mucosa; ammonia
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HYPOCHLORHYDRIA AND INFLAMMATION are hallmarks of early infection with Helicobacter pylori (HP), a pathogenic bacterium that colonizes the gastric antrum and fundus and leads to chronic-active gastritis and peptic ulcer disease. During early infection with HP, it has been suggested that acid secretion may be suppressed by one or more products of HP or by inflammatory mediators that act directly on parietal cells (3, 21). Among the various factors liberated by HP (3), urease may be the most important because it leads to the production of large quantities of ammonia (NH3) that can affect cell function and viability (23, 30, 36, 37, 40). Hypochlorhydria can also occur during chronic renal or hepatic failure or after renal transplantation, when circulating levels of blood NH3 are extremely high (9, 16, 24).
NH3 is a small molecule of 17 Da that acts as a weak base and is at equilibrium with its protonated form (NH4+) at a pKa of 9.233. Thus most of the NH3 produced by HP exists as NH4+ in the acidic environment of the gastric lumen. It has been shown (2) that the apical (or luminal) surface of both gastric parietal and chief cells is relatively impermeable to NH3 and NH4+, suggesting that NH3 formed in the gastric lumen during HP infection would be unable to alter gastric epithelial cell function. However, Yanaka et al. (40) showed that luminal administration of 115 mM ammonium chloride (NH4Cl) at pH 8 (2.7 mM NH3) resulted in a significant decrement in tissue resistance (R) and an increase in H+ back-diffusion that did not occur when the same concentration of NH4Cl was used at pH 5 (3 µM NH3), indicating that luminal NH3 can cause an increase in the paracellular permeability of the gastric mucosa. In addition, it was recently shown (26, 41) that the vacuolating cytotoxin produced in VacA+ HP bacteria increases the paracellular permeability of cultured gastric (GSM06), colonic (T84), and kidney (Madin-Darby canine kidney) cells. Thus both NH3 and vacuolating cytotoxin may facilitate the movement of gastric luminal contents including NH3 to the serosal compartment during infection.
Whereas the apical surface of gastric epithelial cells is impermeable
to NH3, NH3 is freely permeable across the
basolateral cell membrane and NH4+ can enter cells via
cation channels, pumps, or exchange proteins (2).
Basolateral exposure of the bullfrog gastric mucosa to 30 mM
NH4Cl at pH 7.2 (0.47 mM NH3 and 29.53 mM
NH4+) rapidly and completely inhibits acid secretion
and accelerates H+ back-diffusion while it alkalinizes,
induces depolarization, and decreases electrogenic Cl
secretion in bullfrog oxynticopeptic cells (40).
Basolateral exposure of gastric epithelial cells to
NH3/NH4+ also allows these substances to
interact directly with basolateral ion channels, transporters, and
exchangers. Although little is known about the effects of
NH3 and/or NH4+ on basolateral ion
transporters/exchangers in the gastric mucosa, it was suggested by
Yanaka et al. (40) that NH4Cl may block basolateral K+ channel activity. This conclusion was
derived from the observation that high K+ (15 mM) in the
nutrient solution attenuated the decrease in both potential difference
(PD) and R that occurred after exposure to 30 mM nutrient
NH4Cl (40). In addition, partial blockade of K+ channels with 0.2 mM barium (Ba2+)
accelerated the rapid decrease in PD in the presence of
NH4Cl (40). Frog oxyntic cells have
Ca2+-activated, 61-pS, voltage-independent K+
channels that are sensitive to Ba2+ and cAMP-activated,
30-pS, Ca2+-insensitive K+ channels
(20). Other intracellular targets of
NH3/NH4+ may be
Cl/HCO3
exchange (40) or
inhibition of ATP production by mitochondria (37).
Thus the purpose of this study was to determine the mechanism by which
NH4Cl (producing NH3/NH4+)
inhibits acid secretion in the bullfrog gastric mucosa. Using four
different K+-channel blockers, we show that blockade of
basolateral K+-channel activity does not greatly influence
the rate of stimulated acid secretion in the bullfrog gastric mucosa
and thus cannot be the primary site of action of NH4Cl. In
addition, NH4Cl does not inhibit acid secretion by altering
basolateral Na+/H+ exchange,
Na+-K+-2Cl cotransport,
Na+-K+-ATPase,
Cl
/HCO3
exchange, or
Na+-HCO3
cotransport. Furthermore,
NH4Cl does not inhibit acid secretion by neutralizing
secreted H+ in the gastric lumen or by its effects as a
weak base on cytosolic and endosomal pH. We show that, although ATP
depletion occurs in the presence of NH4Cl, the magnitude of
ATP depletion is not sufficient to inhibit stimulated acid secretion.
Our results show that NH4Cl affects the R of
stimulated but not inhibited tissues, suggesting that NH4Cl
specifically targets some aspect of stimulated acid secretion. Because
NH4Cl has been shown to block the activity of
K+ channels in other tissues, we propose that
NH4Cl blocks an apical K+ channel in bullfrog
oxynticopeptic cells. If so, apical K+-channel activity is
important for the regulation of acid secretion in the bullfrog gastric mucosa.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of frog fundic mucosa for Ussing chamber studies:
inhibition and stimulation of acid secretion.
Animals used for this study were maintained in accordance with the
guidelines of the Committee on Animals at the Beth Israel Deaconess
Medical Center and those prepared by the committee on Care and Use of
Laboratory Animals of the National Research Council. Bullfrogs
(Rana catesbeiana) caught in the wild were purchased from
West Jersey (Wenonah, NJ) and kept at room temperature in large water
tanks until use. Stomachs were removed from pithed frogs, and the
fundic mucosa was stripped from underlying external muscle layers and
submucosa to bare the muscularis mucosa as described previously
(10). Stripped mucosae were divided into two halves (one
experimental and one control), each of which was mounted between two
Lucite halves of an Ussing-type chamber with an exposed mucosal area of
0.636 cm2. Mucosal surfaces were bathed with a solution
containing (in mM) 102.4 Na2+, 4.0 K+, 0.8 Mg2+, 1.8 Ca2+, 91.4 Cl, 10.1 SO42
, and 19.3 mannitol and continuously gassed with
100% O2. Mucosal solutions were unbuffered and kept at pH
4.7 with a pH-stat device (Radiometer America, Cleveland, OH). Serosal
surfaces were bathed with (in mM) 102.4 Na2+, 4.0 K+, 0.8 Mg2+, 1.8 Ca2+, 91.4 Cl
, 0.8 SO42
, 0.8 H2PO42
, 10 glucose, and 17.8 HCO32
(pH 7.4) and continuously gassed with 95%
O2-5% CO2.
Electron microscopy. Frog tissues from the Ussing chamber were fixed overnight at 4°C with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), postfixed for 1 h at 4°C with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4), and stained overnight at 4°C with 2% aqueous uranyl acetate. Tissues were dehydrated in graded alcohols and propylene oxide and embedded in LX112 resin. Thin sections, cut parallel to the long axis of gastric glands, were placed on Formvar- and carbon-coated grids and examined with a JEOL 100CX electron microscope.
NH3 and methylamine flux studies. NH3 flux from nutrient to luminal solutions was measured enzymatically by the reductive amination of 2-oxoglutarate to glutamate and reduction of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP in the presence of glutamate dehydrogenase and NH3. In brief, 0.1 ml of nutrient or luminal solution was was mixed with 1.0 ml of NH3 assay solution containing 3.4 mM 2-oxoglutarate and 0.23 mM NADPH in 30 mM Tris buffer (pH 8.0), and the optical density (OD) of the solution was determined at a wavelength of 340 nM (initial OD). After the addition of 0.008 ml of L-glutamate dehydrogenase to each sample, the OD at 340 nM was determined (final OD). The final OD340 was subtracted from the initial OD340, and the concentration of NH3 (not NH3/NH4+) was determined from a standard curve. Oxoglutarate, NADPH, and glutamate dehydrogenase were purchased from Sigma Diagnostics (St. Louis, MO).
For flux studies with NH4Cl, frog gastric mucosae were mounted in Ussing chambers and then stimulated with histamine plus carbachol for 90 min. NH4Cl (10 mM) was added to the nutrient solution, and the nutrient and luminal solutions were assayed for NH3 to establish starting conditions. After 30 min in nutrient 10 mM NH4Cl, samples of both nutrient and luminal solutions were collected and the concentration of NH3 was determined, as described above. For flux studies with methylamine, frog gastric mucosae were mounted in Ussing chambers and one of the paired tissues was stimulated for 90 min with histamine plus carbachol while the other was inhibited with cimetidine (as described above). Methylamine (50 mM), containing 5 µCi of [14C]methylamine, was added to the nutrient solution, and duplicate 0.5-ml samples were taken from the luminal solution every 15 min for 1 h. Samples were mixed with Atomlight scintillation fluid (Packard Instruments, Meriden, CT), and the dpm were counted in a Packard scintillation counter. The concentration of methylamine in the luminal solution was calculated by established techniques.ATP assay. Isolated gastric glands were prepared from the rabbit fundus after high-pressure retrograde aortic perfusion by the technique previously described in detail by Carter et al. (4) as modified from Berglindh and Obrink (1). Isolated glands were maintained in the resting state by the addition of 10 µM cimetidine. Intracellular ATP was measured by the luciferin/luciferase assay in resting glands that were incubated for 30 min at 37°C in the presence or absence of 10 mM NH4Cl (pH 7.4) or 2 mM KCN. ATP was extracted from the glands after precipitation of proteins with 2% TCA and 2 mM EDTA as described previously (38). Bioluminescence was measured in an LKB Wallac model 1250 luminometer (Turku, Finland), and the concentration of ATP was determined with an ATP standard. Results were expressed as micromoles of ATP per milligram of protein, which was determined using the BioRad protein assay.
Statistical analysis. Combined data were expressed as means ± SE. Statistical analyses of data were done with SigmaStat software (Jandel Scientific Software, San Rafael, CA) using the unpaired t-test for analysis of two groups or one-way ANOVA for many groups. Differences were regarded as statistically significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nutrient NH4Cl rapidly and reversibly inhibits
stimulated acid secretion.
Acid secretion in frog fundic mucosa attained a maximal rate of
5.80 ± 0.12 µeq · h1 · cm
2 by 90 min after stimulation (Fig.
1A). While R
remained constant at 164.7 ± 5.61
· cm2 (Fig. 1B), PD declined slowly to 6.00 ± 0.70 mV by 90 min after stimulation (Fig. 1C). Similar
results were obtained after stimulation with forskolin (data not
shown).
|
|
|
Acid secretion is rapidly inhibited by methylamine but not by
imidazole.
Methylamine and imidazole were used to determine whether other small
primary alkylamines or weak bases, respectively, inhibit acid secretion
(Fig. 3). Methylamine inhibited
stimulated acid secretion from 5.81 ± 0.125 to 0.081 ± 0.056 µeq · h1 · cm
2 within
30 min at a minimum concentration of 50 mM (Fig. 3A). However, when methylamine was washed from the nutrient solution, acid
secretion returned to only 60% (3.74 ± 0.41 µeq · h
1 · cm
2) of the starting level
within 60 min (Fig. 3A). When imidazole was used at a
concentration similar to that of NH3 in the nutrient buffer
(100 µM imidazole vs. 92.5 µM NH3), acid secretion
decreased slowly from 5.47 ± 0.26 to 4.86 ± 0.29 µeq
· h
1 · cm
2 in 3 h (180 min).
This reduction in acid secretion over time was not significantly
different from that of control tissues (Fig. 3B). Increasing
the concentration of imidazole 50-fold to 10 mM (5 mM imidazole, 5 mM
imidazole+) decreased acid secretion rapidly from 6.60 ± 0.43 to 4.60 ± 0.43 µeq · h
1 · cm
2 in 30 min and to 4.06 ± 0.46 µeq · h
1 · cm
2 in 3 h (data not
shown). When 10 mM nutrient NH4Cl was added to
imidazole-treated tissues, acid secretion was inhibited to 0 ± 0 µeq · h
1 · cm
2 within 30 min (Fig. 3B).
|
Serosal-to-mucosal flux of NH3 and methylamine is not great enough to neutralize secreted H+. To determine whether nutrient 10 mM NH4Cl or 50 mM methylamine blocks acid secretion by moving rapidly from nutrient to luminal solutions, and thus neutralizing secreted H+, the flux of NH3 and methylamine was measured.
After addition of 10 mM NH4Cl to the nutrient solution, the NH3 concentration was 599.76 µM. At the start, NH3 was not detectable in the luminal solution. By 30 min after addition of 10 mM NH4Cl to the nutrient solution, the nutrient NH3 concentration was reduced to 538.3 ± 6.7 µM. Thus 61.46 µM NH3 was lost from the nutrient solution. However, luminal NH3 concentration after 30 min increased in only one group (n = 4), resulting in a mean concentration of 0.00133 ± 0.00133 µM. These results suggest that the NH3 lost from the nutrient buffer within 30 min resides in the cells and intracellular spaces of the tissue. It is possible, however, that NH3 cannot be detected in the luminal solution of stimulated tissues because the forward movement of NH3 is balanced by the backward movement of NH4+ into the cell by H+-K+-ATPase, as has been described previously (8). Thus serosal-to-mucosal flux studies with methylamine, a compound that should not interact with H+-K+-ATPase, were performed (Fig. 4).
|
Blockade of basolateral K+-channel activity does not
mitigate the inhibitory effects of NH4Cl on stimulated acid
secretion.
Ba2+ at 0.5 and 1 mM reduced acid secretion in 3 h
(180 min) from 5.23 ± 0.29 and 5.35 ± 0.22 µeq · h1 · cm
2 to 4.12 ± 0.38 and
4.20 ± 0.21 µeq · h
1 · cm
2, respectively (Fig.
5A). This decline in
the rate of acid secretion over time was not significantly different
from that of control tissues (Fig. 5A). In contrast,
incubation of stimulated tissues with 5 mM Ba2+ decreased
the rate of acid secretion from 5.19 ± 0.22 to 2.89 ± 0.33 µeq · h
1 · cm
2 in 3 h
(180 min). This reduction in the rate of acid secretion was
significantly different from that of control tissues (Fig. 5A). Acid secretion in the presence of another wide-spectrum
K+-channel blocker such as TEA (Fig.
6A), tolbutamide, which blocks ATP-dependent K+-channel activity (Fig. 6B), or
clotrimazole, which blocks Ca2+- and cAMP-dependent
K+-channel activity (Fig. 6B), showed no
significant decline compared with control tissues. When tissues treated
with 0.5-5 mM Ba2+, 1-10 mM TEA, 10-100 µM
tolbutamide, or 10 µM clotrimazole were incubated with 10 mM
NH4Cl, acid secretion was inhibited to 0 µeq · h
1 · cm
2 within 30 min in all groups
(Figs. 5 and 6).
|
|
Blockade of basolateral transporter activity does not significantly
abrogate NH3-induced inhibition of acid secretion.
Incubation of stimulated tissues with 100 µM bumetanide, 1 mM
amiloride, or 1 mM ouabain for 60 min caused no significant reduction
in acid secretion (Figs.
7A). In contrast, acid
secretion was reduced from 5.25 ± 0.12 to 2.29 ± 0.18 µeq · h1 · cm
2 in
stimulated tissues incubated with 0.3 mM DIDS for 60 min (Fig. 7B). When stimulated tissues were incubated for 30 min with
one of the four ion transport blockers and then further incubated with
10 mM NH4Cl, acid secretion declined to 0.645 ± 0.278, 0.188 ± 0.140, 0, and 0 µeq · h
1 · cm
2 for bumetanide, amiloride,
ouabain, and DIDS, respectively, within 30 min (Fig. 7). Similar
results were obtained when a combination of bumetanide, amiloride, and
oubain (followed by NH4Cl) was added to stimulated tissues
(data not shown).
|
KCN inhibits stimulated acid secretion similarly to 10 mM
NH4Cl but results in significantly greater depletion of
intracellular ATP.
Acid secretion in the frog gastric mucosa was inhibited in a
dose-dependent manner after addition of the metabolic inhibitor potassium cyanide (KCN). Whereas 250 µM KCN significantly reduced acid secretion (data not shown), 2 mM KCN was required to inhibit acid
secretion to 0.33 ± 0.04 µeq · h1 · cm
2 within 30 min (Fig.
8A). The rate of
reduction of acid secretion with 2 mM KCN was similar to that of 10 mM
NH4Cl and three times faster than with cimetidine or
omeprazole (Table 1). Inhibition of acid secretion with 2 mM KCN was
accompanied by an increase in R from 112.4 ± 8.16 to
613.7 ± 38.19
· cm2 and PD from 4.72 ± 2.59 to 15.05 ± 1.20 mV (data not shown).
|
NH4Cl does not increase R of cimetidine-inhibited
tissues.
Tissues were incubated with cimetidine until acid secretion reached 0 µeq · h1 · cm
2; experiments
were performed only if the R of paired tissues were different by 10% or less. When 10 mM NH4Cl was added to
the nutrient solution of cimetidine-inhibited tissues (pH 7.2), there
was no change in R compared with control tissues incubated
with NaCl (Fig. 9). These results are in
contrast to those in tissues stimulated with histamine plus carbachol
(Fig. 1B), where tissue R increased significantly
in the presence of 10 mM NH4Cl. In addition, when K+-channel activity in cimetidine-inhibited tissues was
blocked with either 1 or 5 mM Ba2+, R increased
to 126.0 ± 5.0% and 140.0 ± 7.0%, respectively, of the
initial R (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study shows that basolateral exposure of gastric oxyntic cells to as little as 92.5 µM NH3, present in 10 mM NH4Cl at pH 7.2, completely inhibits acid secretion within 30 min. Similar concentrations of NH3 could permeate the gastric mucosa during acute infection with HP through defects in the epithelium or circulate in the blood of patients with chronic renal failure or hepatic disease. This inhibition of acid secretion with NH4Cl is more than three times faster than with other acid inhibitors such as cimetidine (H2 receptor agonist) or omeprazole (proton pump inhibitor). Because NH4Cl inhibits acid secretion after stimulation with histamine plus carbachol or forskolin, it is likely that NH4Cl acts at a site distal to H2 receptor binding and to the activation of adenylate cyclase in the cAMP-signaling cascade. Inhibition of acid secretion by NH4Cl was accompanied by an increase in PD and R nearly identical in magnitude to the increase in PD and R that occurred during inhibition of acid secretion by either cimetidine or omeprazole.
Our results using 10 mM NH4Cl to inhibit acid secretion are consistent with those of Yanaka et al. (40) who showed that 30 mM nutrient NH4Cl (270 µM NH3) completely inhibits stimulated acid secretion in the frog. Whereas the NH4Cl-induced inhibition of acid secretion in the present study was completely reversed after withdrawal of NH4Cl, Yanaka et al. (40) showed only minimal recovery after withdrawal of NH4Cl. These results suggest that the ability of acid secretion to recover fully after an acute challenge with NH4Cl is concentration dependent. The finding that the morphology of oxyntic cells remained stimulated despite the absence of acid secretion accounts for the ability of these cells to regain nearly normal rates of acid secretion rapidly after the withdrawal of NH4Cl from the nutrient solution; reassembly of the apical surface takes nearly 2 h from the inhibited configuration in frog gastric mucosa (10).
The way in which NH3 or NH4+ enters the
basolateral membrane of gastric oxyntic cells is not known. However,
the basolateral membrane of gastric oxyntic cells possess multiple
permeability pathways for Na+ and K+
(Na+/H+ exchange,
Na+-HCO3 cotransport,
Na+-K+-2Cl
cotransport,
Na+-K+-ATPase) that could transport
NH4+ into the cell. Our results demonstrate, however,
that basolateral ion transporters do not facilitate the rapid movement
of NH4+ into gastric oxyntic cells because blockade of
basolateral ion transport activity did not mitigate the effect of
NH4Cl on acid secretion. It is most likely that
NH3 enters cells at the basolateral membrane by passive
diffusion as was postulated by Yanaka et al. (40) and then
is rapidly converted to NH4+ in the cytoplasm at
neutral pH. The results presented here differ from those obtained in
the kidney where NH4+ is moved rapidly into cells by
renal Na+-K+-ATPase or by
Na+-K+-2Cl
cotransporter activity
depending on cell type (15).
Several lines of evidence from the present study suggest that
NH4Cl does not block acid secretion by blockade of
basolateral ion transport activity. We show that inhibition of
Na+/H+ exchange with amiloride,
Na+-K+-ATPase with ouabain, or
Na+-K+-2Cl cotransport with
bumetanide for 30 min has little effect on stimulated acid secretion in
the frog gastric mucosa. These results are consistent with other
reports (11, 28) in which blockade of
transport activity had little effect on stimulated acid secretion in
the frog. Thus even if NH4Cl inhibited the activity of one
of these three exchangers, there would be no demonstrable effect on
acid secretion. In contrast, our results show that acid secretion in the frog gastric mucosa was partially, but not completely, inhibited by
blockade of Na+-HCO3
cotransport and/or
Cl
/HCO3
exchange with DIDS. Similar
results were obtained with DIDS in the rat, mouse, and rabbit gastric
mucosa (11, 12, 22). The finding
that acid secretion can be inhibited by blockade of Na+-HCO3
cotransport and/or
Cl
/HCO3
exchange suggests that
NH4Cl could reduce acid secretion, in part, by blockade of
one or both of these basolateral transporters. Against this possibility
is the finding that the addition of NH4Cl to tissues
incubated with DIDS immediately reduced acid secretion to 0, suggesting
that NH4Cl and DIDS act at different sites. Thus it is
unlikely that NH4Cl inhibits acid secretion by blocking Na+-HCO3
cotransport and/or
Cl
/HCO3
exchange in gastric oxyntic cells.
Imidazole can be used to mimic the effects of NH3 as a weak
base. Like NH3, an equimolar concentration of imidazole
should result in a similar amount of cytosolic alkalinization and
neutralization of acidic intracellular compartments due to its ability
to easily permeate cell membranes (19). However, when acid
secretion was measured for 3 h in the presence of 200 µM
nutrient imidazole at pH 7.1 (100 µM imidazole vs. 92.3 µM
NH3), the rate of acid secretion was not significantly
different from that of control tissues. Furthermore, acid secretion in
3 h was inhibited only slightly from 6.6 to 4.03 µeq · h1 · cm
2 in the presence of 10 mM
nutrient imidazole at pH 7.1 (5 mM imidazole). The finding that 10 mM
nutrient NH4Cl inhibited acid secretion immediately in the
presence of either 200 µM or 5 mM imidazole at pH 7.1 suggests that
the action of NH3 and imidazole are different. A similar
conclusion was drawn from studies using NH4Cl, imidazole, and other weak bases in intestinal T84 cells (27).
Methylamine, a C1 primary alkylamine that is larger and
more lipophilic than NH3 (C0 primary
alkylamine), can be used to test the effects of related primary
alkylamines on acid secretion. Our results clearly demonstrate that
methylamine rapidly inhibits acid secretion in the frog gastric mucosa.
Because of the larger size and lipophilicity, a larger concentration of
methylamine was required to inhibit acid secretion as effectively as
with NH4Cl. In addition, acid secretion did not recover
fully after inhibition with methylamine because methylamine will not
exit cells as rapidly as does NH4Cl. Our results are
consistent with those by Hrnjez et al. (14), who showed
that related primary alkylamines including C0 (ammonium) to
C8 (octylamine) inhibited stimulated Cl
secretion in intestinal T84 cells. Because alkylamines were shown to
inhibit Cl
secretion by blocking resting basolateral
K+-channel activity (14), we considered the
possibility that NH4Cl and methylamine inhibit
K+-channel activity in gastric oxyntic cells.
Our data show, however, that exposure of the gastric mucosa to NH4Cl does not block acid secretion by blockade of basolateral K+-channel activity. From the data presented here and by others (18, 25, 33-35), it is clear that blockade of basolateral K+-channel activity does not significantly influence the rate of stimulated acid secretion in the gastric mucosa. The present study shows that 1 h or more of incubation with Ba2+ (5 mM) is required to significantly reduce the rate of stimulated acid secretion in the frog gastric mucosa, and by 3 h acid secretion was reduced only 44% compared with control tissues. In addition, inhibition of acid secretion does not occur in 3 h when the Ba2+ concentration is reduced to 0.5 or 1 mM. These results are consistent with those reported by Schwartz et al. (35) and McLennan et al. (18), who incubated the gastric mucosa for 10-20 min in Ba2+ and saw little effect on stimulated acid secretion at concentrations <5 mM. Incubation of stimulated tissues with Ba2+ also caused a significant increase in R with little to no influence on PD. These results are consistent with those previously reported in both frog and piglet mucosa (18, 25, 33-35). Inhibition of K+-channel activity with tolbutamide, clotrimazole, or TEA had no effect on acid secretion, R, or PD, suggesting that these K+-channel blockers are not effective in the frog gastric mucosa. Similar conclusions were reported recently (5) for tolbutamide in rabbit gastric glands.
The finding that an increase in R of the gastric mucosa incubated with NH4Cl occurs only in stimulated tissues suggests that NH4Cl acts to specifically inhibit some aspect of stimulated acid secretion. In addition to numerous basolateral K+ channels that recycle K+ imported by Na+-K+-ATPase, it is thought that oxyntic and parietal cells possess an apical K+ channel(s) that is (are) active only during acid secretion. The apical K+ channel facilitates the movement of cytoplasmic K+ into the gastric lumen; K+ is then recycled back into the cell by H+-K+-ATPase activity during acid secretion (7). In a preliminary report by Horio et al. (13), it was determined that the apical membrane of stimulated parietal cells has an inwardly rectifying K+ channel (Kir4.1). We propose that it is this K+ channel that is blocked by NH4Cl and methylamine. If so, the apical K+ channel represents an important regulatory site for activity of gastric H+-K+-ATPase. Interestingly, it has been shown (6) that NH4+ is a potent inhibitor of Ca2+-dependent inwardly rectifying K+-channel activity in HeLa cells. Studies that address the relationship of NH4+ and apical K+-channel activity in gastric oxyntic and parietal cells are important and merit further investigation.
If an apical K+ channel is blocked by NH4Cl, it is important to explain 1) why only NH3 and not other K+-channel blockers inhibit acid secretion and 2) why NH3 and Ba2+ do not effectively block acid secretion when administered from the luminal solution. We believe it is likely that NH4Cl blocks acid secretion from the cytosolic face of the K+ channel as was described for inhibition (by NH4Cl) of the inwardly rectifying K+ channel in HeLa cells (6). Thus NH4Cl can get into cells and to the cytosolic aspect of the apical membrane by virtue of its lipophilicity but Ba2+ (and other K+channel blockers) cannot. This hypothesis is consistent with studies (29, 39) that showed isolated (apical) membrane vesicles have a Ba2+-inhibitable K+ conductance whereas luminal Ba2+ (up to 20 mM) had no effect on acid secretion in the intact frog gastric mucosa (35). These studies suggest that Ba2+ can effectively block an apical K+ conductance when the apical membrane is isolated from the intact cell. It is not clear to us why NH4Cl does not inhibit acid secretion from the apical surface. It may be due to the lack of permeability of the apical membrane to NH4Cl (2) or may be because the secretory "flush" during acid secretion makes it difficult for substances to interact directly with the apical membrane. Further work is needed to understand, in detail, the effects of NH4Cl on acid secretion in intact tissues.
Our hypothesis concerning the action of NH3 on gastric oxyntic cells is not consistent with that set forth by Fryklund et al. (8). In that study (8), aminopyrine uptake, oxygen consumption, and glucose oxidation were shown to increase in isolated and stimulated glands (rabbit) and decrease in inhibited glands. Because glucose oxidation also increased in the presence of NH4+, it was suggested that NH3 stimulates H+-K+-ATPase activity. It was proposed that NH3 enters the canalicular membrane and is protonated by secreted H+ to form NH4+, and then NH4+ acts as a surrogate for K+ on the active H+-K+-ATPase. We cannot rule out the possibility that titration of secreted H+ by NH3 and recycling of NH4+ is the mechanism by which NH4Cl inhibits acid secretion. However, because the flux of a related primary alkylamine (methylamine) is much slower than the measured rate of acid secretion in stimulated tissues, it seems unlikely that titration and recycling can account for a rapid and complete inhibition of acid secretion in gastric tissues. Because of the effect of NH4Cl on R in inhibited and stimulated tissues and the inhibition of acid secretion by related alkylamines that do not interact with H+-K+-ATPase, we believe that the K+ channel hypothesis warrants further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. William Silen, Jeffrey Matthews, David Soybel, Sylvana Curci, and Aldebaran Hofer for helpful discussions concerning the results of this manuscript. We also thank Drs. William Silen, John Forte, and Jeffrey Matthews for critical reading of the manuscript. The technical assistance of Rudite Jansons is gratefully acknowledged.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-15681 (S. J. Hagen) and Harvard Digestive Diseases Center Grant DK-34854.
Address for reprint requests and other correspondence: S. J. Hagen, Dept. of Surgery, Beth Israel Deaconess Medical Center, RW 863, 330 Brookline Ave. Boston, MA 02215 (E-mail: shagen{at}caregroup.harvard.edu).
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 15 December 1999; accepted in final form 12 March 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berglindh, T,
and
Obrink KJ.
A method for preparing isolated glands from the rabbit gastric mucosa.
Acta Physiol Scand
96:
150-159,
1976[ISI][Medline].
2.
Boron, WF,
Waisbren SJ,
Bodlin IM,
and
Geibel JP.
Unique permeability barrier of the apical surface of parietal and chief cells in isolated perfused gastric glands.
J Exp Biol
196:
347-360,
1994
3.
Calam, J,
Gibbons A,
Healey ZV,
Bliss P,
and
Arebi N.
How does Helicobacter pylori cause mucosal damage? Its effect on acid and gastric physiology.
Gastroenterology
113, Suppl:
S43-S49,
1997[ISI][Medline].
4.
Carter, KJ,
Lee HH,
Goddard PJ,
Yanaka A,
Paimela H,
and
Silen W.
Cell survival in rabbit gastric glands: effect of extracellular pH, osmolarity, and anoxia.
Am J Physiol Gastrointest Liver Physiol
265:
G379-G387,
1993
5.
Del Valle, JC,
Olea J,
Pereda C,
Gutierrez Y,
Feliu JE,
and
Rossi I.
Sulfonylurea effects on acid and pepsinogen secretion in isolated rabbit gastric glands.
Eur J Pharmacol
343:
225-232,
1998[ISI][Medline].
6.
Diaz, M,
Riquelme G,
and
Sepulveda FV.
Ammonium inhibition of Ca2+-dependent inwardly rectifying K+ currents in HeLa cells.
Biochim Biophys Acta
1284:
119-121,
1996[ISI][Medline].
7.
Forte, JG,
and
Reenstra WW.
Chloride transport by gastric mucosa.
In: Advances in Comparative and Environmental Physiology. Berlin: Springer-Verlag, 1994, chapt. 13, p. 239-259.
8.
Fryklund, J,
Gedda K,
Scott D,
Sachs G,
and
Wallmark B.
Coupling of H+-K+-ATPase activity and glucose oxidation in gastric glands.
Am J Physiol Gastrointest Liver Physiol
258:
G719-G727,
1990
9.
Gingell, JC,
Burns GP,
and
Chisholm GD.
Gastric acid secretion in chronic uraemia and after renal transplantation.
Br Med J
4:
424-426,
1968[ISI][Medline].
10.
Hagen, SJ,
Yanaka A,
and
Jansons R.
Localization of brush border cytoskeletal proteins in gastric oxynticopeptic cells from the bullfrog Rana catesbeiana.
Cell Tissue Res
275:
255-267,
1994[ISI][Medline].
11.
Horie, S,
Yano S,
and
Watanabe K.
Effects of drugs acting on Cl-HCO3
and Na+-H+ exchangers on acid secretion in the rat gastric mucosa sheet preparation.
Eur J Pharmacol
229:
15-19,
1992[ISI][Medline].
12.
Horie, S,
Yano S,
and
Watanabe K.
Inhibition of gastric acid secretion in vivo and in vitro by an inhibitor of Cl-HCO3
exchanger, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
J Pharmacol Exp Ther
265:
1313-1318,
1993[Abstract].
13.
Horio, Y,
Mouri T,
Fujita A,
Inanobe A,
Tanemoto M,
and
Kurachi Y.
Inwardly rectifying K+-channel Kir4.1 participates in gastric acid secretion (Abstract).
Physiologist
42:
A18,
1999.
14.
Hrnjez, BJ,
Song JC,
Mayol JM,
and
Matthews JB.
Ammonia inhibits intestinal Cl secretion via ammonium block of basolateral K+ channels (Abstract).
Gastroenterology
112:
A370,
1997[ISI].
15.
Knepper, MA,
Packer R,
and
Good DW.
Ammonium transport in the kidney.
Physiol Rev
69:
179-249,
1989
16.
Mathur, KP,
and
Agrawal SP.
A study of the acid gastric secretion in portal hypertension.
J Indian Med Assoc
60:
288-291,
1973[Medline].
17.
Mayol, JM,
Hrnjez BJ,
Akbarali HI,
Song JC,
Smith JA,
and
Matthews JB.
Ammonia effect on calcium-activated chloride secretion in T84 intestinal epithelial monolayers.
Am J Physiol Cell Physiol
273:
C634-C642,
1997
18.
McLennan, WL,
Machen TE,
and
Zeuthen T.
Ba2+ inhibition of electrogenic Cl secretion in vitro frog and piglet gastric mucosa.
Am J Physiol Gastrointest Liver Physiol
239:
G151-G160,
1980
19.
Megraud, F,
Neman-Simha V,
and
Brugmann D.
Further evidence of the toxic effect of ammonia produced by Helicobacter pylori urease on human epithelial cells.
Infect Immun
60:
1858-1863,
1992[Abstract].
20.
Mieno, H,
Komatsu H,
Harada W,
Katayama N,
Shirakawa T,
Inoue M,
and
Kajiyama G.
Regulation of Ca2+-activated K+ channel activity in bullfrog oxyntic cells.
J Gastroenterol
29, Suppl7:
55-58,
1994[ISI][Medline].
21.
Mobley, HLT
Helicobacter pylori factors associated with disease development.
Gastroenterology
113, Suppl:
S21-S28,
1997[ISI][Medline].
22.
Muallem, S,
Blissard D,
Cragoe EJ,
and
Sachs G.
Activation of the Na+/H+ and Cl/HCO3
exchange by stimulation of acid secretion in the parietal cell.
J Biol Chem
263:
14703-14711,
1988
23.
Murakami, M,
Saita H,
Teramura S,
Dekigai H,
Asagoe K,
Kusaka S,
and
Kita T.
Gastric ammonia has a potent ulcerogenic action on the rat stomach.
Gastroenterology
105:
1710-1715,
1993[ISI][Medline].
24.
Muto, S,
Murayama N,
Asano Y,
Hosoda S,
and
Miyata M.
Hypergastrinemia and achlorhydria in chronic renal failure.
Nephron
40:
143-148,
1985[ISI][Medline].
25.
Pacifico, AD,
Schwartz M,
MacKrell TN,
Spangler SG,
Sanders SS,
and
Rehm WS.
Reversal by potassium of an effect of barium on the frog gastric mucosa.
Am J Physiol
216:
536-541,
1969[ISI][Medline].
26.
Papini, E,
Satin B,
Norais N,
DeBernard M,
Telford JL,
and
Rappuoli R.
Selective increase of the permeability of polarized epithelial monolayers by Helicobacter pylori vacuolating cytotoxin.
J Clin Invest
102:
813-820,
1998
27.
Prasad, M,
Smith JA,
Resnick A,
Awtrey CS,
Hrnjez BJ,
and
Matthews JB.
Ammonia inhibits cAMP-regulated intestinal Cl transport: asymmetric effects of apical and basolateral exposure and implications for epithelial barrier function.
J Clin Invest
96:
2142-2151,
1995[ISI][Medline].
28.
Reenstra, WW,
Bettencourt JD,
and
Forte JG.
Mechanisms of active Cl secretion by frog gastric mucosa.
Am J Physiol Gastrointest Liver Physiol
252:
G543-G547,
1987
29.
Reenstra, WW,
and
Forte JG.
Characterization of K+ and Cl conductances in apical membrane vesicles from stimulated rabbit oxyntic cells.
Am J Physiol Gastrointest Liver Physiol
259:
G850-G858,
1990
30.
Ricci, V,
Sommi P,
Fiocca R,
Cova E,
Figura N,
Romano R,
Ivey KJ,
Solcia E,
and
Ventura U.
Cytotoxicity of Helicobacter pylori on human gastric epithelial cells in vitro: role of cytotoxin(s) and ammonia.
Eur J Gastroenterol Hepatol
5:
687-694,
1993[ISI].
31.
Rufo, PA,
Jiang L,
Moe SJ,
Brugnara C,
Alper SL,
and
Lencer WI.
The antifungal antibiotic, clotrimazole, inhibits Cl secretion by polarized monolayers of human colonic epithelial cells.
J Clin Invest
98:
2066-2075,
1996
32.
Rufo, PA,
Merlin D,
Riegler M,
Ferguson-Maltzman MH,
Dickinson BL,
Brugnara C,
Alper SL,
and
Lencer WI.
The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances.
J Clin Invest
100:
3111-3120,
1997
33.
Sanders, SS,
Noyes DH,
Spangler SG,
and
Rehm WS.
Demonstration of a barium-potassium antagonism on lumen side of in vitro frog stomach.
Am J Physiol
224:
1254-1259,
1973[ISI][Medline].
34.
Sanders, SS,
Shoemaker RL,
and
Rehm WS.
Electrogenic HCl theory in light of effects of SCN and Ba2+ on frog stomach.
Am J Physiol Endocrinol Metab Gastrointest Physiol
234:
E298-E307,
1977.
35.
Schwartz, M,
Pacifico AD,
MacKrell TN,
Jacobson A,
and
Rehm WS.
Effects of barium on the in vitro frog gastric mucosa.
Proc Soc Exp Biol Med
127:
223-225,
1968.
36.
Triebling, AT,
Korsten MA,
Dlugosz JW,
Paronetto F,
and
Lieber CS.
Severity of Helicobacter-induced gastric injury correlates with gastric juice ammonia.
Dig Dis Sci
36:
1089-1096,
1991[ISI][Medline].
37.
Tsujii, M,
Kwano S,
Tsuji S,
Fusamoto H,
Kamada T,
and
Sato N.
Mechanism of gastric mucosal damage induced by ammonia.
Gastroenterology
102:
1881-1888,
1992[ISI][Medline].
38.
Unno, N,
Menconi MJ,
Salzman AL,
Smith M,
Hagen SJ,
Ge Y,
Ezzell RM,
and
Fink MP.
Hyperpermeability and ATP depletion induced by chronic hypoxia or glycolytic inhibition in Caco-2BBe monolayers.
Am J Physiol Gastrointest Liver Physiol
270:
G1010-G1021,
1996
39.
Wolosin, JM,
and
Forte JG.
K+ and Cl conductances in the apical membrane from secreting oxyntic cells are concurrently inhibited by divalent cations.
J Membr Biol
83:
261-272,
1985[ISI][Medline].
40.
Yanaka, A,
Muto H,
Ito S,
and
Silen W.
Effects of ammonium ion and ammonia on function and morphology of in vitro frog gastric mucosa.
Am J Physiol Gastrointest Liver Physiol
265:
G277-G288,
1993
41.
Yanaka, A,
Suzuki H,
Matsui H,
Nakahara A,
Tanaka N,
and
Muto H.
Helicobacter pylori vacuolating cytotoxin (Vac A) increases paracellular permeability across the gastric surface mucous cell monolayers (Abstract).
Gastroenterology
116:
A362,
1999.