1 Department of Biochemistry, Faculty of Medicine, and Departments of 2 Experimental Endocrinology and 3 Gastroenterology, Clínica Puerta de Hierro, Universidad Autónoma de Madrid, E-28029 Madrid, Spain
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ABSTRACT |
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Ethanol (1-20% vol/vol) caused a dose-dependent reduction
in the basal rate of acid formation in isolated rabbit gastric glands with a calculated EC50 value of 4.5 ± 0.2%. Ethanol
also reduced ATP levels in isolated gastric glands and in cultured
parietal cells (EC50: 8.8 ± 0.4% and 8.5 ± 0.2%, respectively) and decreased both basal and forskolin-stimulated
cAMP levels. In studies carried out in gastric gland microsomes,
ethanol inhibited the hydrolytic activity of
H+-K+-ATPase (EC50: 8.5 ± 0.6%), increased passive proton permeability (EC50:
7.9%), and reduced H+-K+-ATPase-dependent
proton transport (EC50: 3%). Our results show that the
inhibition of gastric acid secretion observed at low concentrations of
ethanol (5%) is mainly caused by the specific impairment of
H+-K+-ATPase-dependent proton transport across
cell membranes rather than inhibition of the hydrolytic activity of
H+-K+-ATPase, reduction in the cellular content
of ATP, or increase in the passive permeability of membranes to
protons, although these changes, in combination, must be relevant at
concentrations of ethanol
7%.
gastric glands; proton permeability; parietal cells
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INTRODUCTION |
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IN VIVO AND IN VITRO
EXPERIMENTS have shown that ethanol exerts a dose-related
biphasic effect on gastric acid secretion in humans and in different
animal models (11, 30, 33). Thus, in humans, low topical
concentrations of ethanol (1.4-4% vol/vol) have been shown to
moderately stimulate acid production, whereas concentrations of
5-40% have no effect or, rather, an inhibitory effect
(33). Similarly, in experiments carried out in isolated toad gastric mucosa, ethanol applied at low concentrations (2%-10% vol/vol) to either the luminal or the submucosal side was a potent stimulator of HCl secretion, whereas high concentrations (20%) were
inhibitory (11). Furthermore, in isolated rabbit gastric glands stimulated by histamine, low concentrations of ethanol (0.2%-5% vol/vol) significantly potentiated the rate of acid
formation as well as the glandular concentration of cAMP, whereas the
presence of 10% ethanol markedly decreased both HCl secretion and cAMP levels (30).
Different experimental evidence suggests that the rise in gastric acid secretion in vivo induced by low concentrations of ethanol may be caused by its stimulation of the release of humoral agents such as histamine (2, 16, 28) or gastrin (6, 17). Moreover, because ethanol has a fluidizing effect on membrane phospholipids in intact cell systems (20), a specific potentiation of the interaction between histamine receptor, activated by endogenous histamine, and parietal cell adenylate cyclase has also been implicated in the stimulatory effects of low ethanol concentrations on gastric acid secretion in vivo (30).
On the other hand, the mechanisms by which ethanol impairs gastric acid secretion have been related to a variety of cellular processes involved in stimulus-secretion coupling. Possible targets of ethanol toxicity that may impair the secretory function of the parietal cell are, among others, the membrane-bound histamine receptor, the adenylate cyclase complex, polymerization of actin and fusion of cytosolic microsomes with the apical membrane, H+-K+-ATPase activity, membrane permeability to certain ions, and cell energy charge (26, 30).
In this work, we have carried out a systematic study on the influence
of different concentrations of ethanol (1-20% vol/vol) on the
rate of acid formation in isolated rabbit gastric glands. This in vitro
model allows us to investigate direct effects of ethanol on gastric
acid formation in the absence of neural or endogenous hormonal
influences. The accompanying changes in gastric gland cell viability,
ATP and cAMP levels, as well as the effect of ethanol on
H+-K+-ATPase activity and on both passive and
H+-K+-ATPase-dependent transport of protons
across microsomal membranes, have also been investigated. Our results
show that 1% (vol/vol) ethanol did not significantly affect the rates
of basal and forskolin-stimulated acid formation in isolated rabbit
gastric glands. In contrast, concentrations of ethanol 5% caused a
marked reduction in the rate of acid production in both nonstimulated
and forskolin-treated gastric glands, inhibition being almost complete
at 10% ethanol. Although ethanol caused a dose-dependent reduction in
both ATP and cAMP levels in gastric glands and parietal cells,
inhibited the hydrolytic activity of
H+-K+-ATPase, and increased the passive proton
permeability of microsomal membranes, the inhibition of the rate of
acid formation elicited by low concentrations of ethanol (<5%) seemed
to be mainly related to an impairment of the
H+-K+-ATPase-dependent transport of protons
across the cell membranes.
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MATERIALS AND METHODS |
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Animals. Male rabbits (New Zealand White, 1.5-2.5 kg body wt) from our inbred colony were used. The animals were fed on a standard chow (N-25; Moragón, Toledo, Spain) and water ad libitum and were housed in animal quarters at constant temperature (23°C) with a fixed (12 h) light cycle. The animal experimentation described was conducted in accordance with the highest standards of humane animal care. All animals were anesthetized with pentobarbital sodium (30 mg/kg body wt iv) immediately before the experiments.
Reagents. Collagenase A from Clostridium histolyticum (type I), forskolin, IBMX, N,N'-dicyclohexylcarbodiimide (DCCD), ouabain, oligomycin, nigericin, valinomycin, and acridine orange were purchased from Sigma (St. Louis, MO). [Dimethyl-amine-14C]aminopyrine (100-120 mCi/mmol) and the cAMP 125I-radioimmunoassay kit were obtained from Amersham International (Little Chalfont, UK). Matrigel was purchased from Becton Dickinson (Bedford, MA). Percoll was obtained from Pharmacia Biotech (Uppsala, Sweden). Ethanol was from Merck (Darmstadt, Germany). The remaining reagents, all of analytical grade, were from Boehringer Mannheim (Mannheim, Germany), Sigma, or Merck.
Gastric gland isolation. Gastric glands were prepared according to the method reported by Berglindh and Öbrink (8) as previously described by Rossi et al. (31). Briefly, the stomach of an anesthetized rabbit was perfused in situ with phosphate-buffered saline (in mM: 149.6 NaCl, 3 K2HPO4 and 0.64 NaH2PO4, pH 7.4) at 37°C. The mucosa from the gastric corpus was freed from the muscle layers, minced, and incubated with collagenase (0.5 mg/ml) at 37°C for 30-45 min. The resulting digest was filtered through a nylon mesh, rinsed three times, and resuspended at a final concentration of 2-5 mg dry weight/ml in medium A (in mM: 132.4 NaCl, 5.4 KCl, 5 Na2HPO4, 1 NaH2PO4, 1.2 MgSO4, 1 CaCl2, 15 HEPES, and 10 glucose with 10 mg/l phenol red and 1 mg/ml bovine serum albumin, at pH 7.3). The incubations were performed with agitation (100 strokes/min) at 37°C in an 95% O2-5% CO2 atmosphere. Isolated rabbit gastric glands had the typical rodlike appearance (0.3-0.7 nm in length). More than 98% of the different cells present in the glands excluded trypan blue dye (0.1% wt/vol). The dry weight of the gastric gland suspensions was calculated in 1-ml aliquots; after centrifugation (10,000 g for 3 min), the pellets were dried overnight at 60°C and then weighed.
Gastric parietal cell isolation. Rabbit gastric parietal cells were isolated according to the method reported by Beales and Calam (5). Perfusion of the stomach and isolation of the gastric mucosa was carried out as described in Gastric gland isolation. After the mucosa was minced into small fragments, it was subjected to sequential digestion by collagenase (0.35 mg/ml) and EDTA as previously reported (5) to obtain a crude suspension of isolated gastric cells. Parietal cells were enriched from the crude cell suspension by centrifugal elutriation in a Beckman J6-MC elutriator using a Beckman JE 5.0 rotor (5). Further purification of the parietal cells was performed by density gradient centrifugation in 30% Percoll (20 min at 2,000 g). The resulting top cell layer, which contained >90% purified parietal cells (by Giemsa staining), was recovered, washed, and resuspended in culture medium (1:1 mixture of Ham's F12-DMEM supplemented with 15 mM HEPES pH 7.4, 7% fetal bovine serum, 100 µg/ml gentamicin sulfate, and 8 µg/ml insulin). Parietal cells were plated (0.4 million cells/well) on 12-well cell culture clusters (Corning Costar, Cambridge, MA) coated with Matrigel (1:9 in sterile water). The cultured cells were maintained in a 95% O2-5% CO2 atmosphere at 37°C for 24 h.
Measurement of acid production. Acid secretion by isolated gastric glands was determined by accumulation of the weak base [14C]aminopyrine in the canalicular compartment, following the method described by Berglindh et al. (7) with minor modifications. Thus 1.5-ml samples of the gland suspensions were incubated in medium A, as indicated in Gastric gland isolation, in the presence of 0.88 µM [14C]aminopyrine (0.1 µCi/ml) and different concentrations of ethanol with or without 1 µM forskolin. After 30-min incubation, 1-ml aliquots of the gland suspensions were taken and immediately centrifuged (10,000 g for 20 s). The supernatants were quickly removed, and the pellets were rinsed twice with medium A, dried, and dissolved in 250 µl of 60% HNO3 at 50°C for 15 min. The radioactivity of the supernatants and those of acid extracts were counted in a liquid scintillation spectrometer. Results are expressed as the ratio of intraglandular (acid space) to extraglandular [14C]aminopyrine concentration, which was calculated as described elsewhere (7). Correction for the [14C]aminopyrine trapped in the pellet was performed by incubating glands in the presence of 10 mM thiocyanate (12).
Preparation of gastric gland microsomes. Nonstimulated rabbit gastric glands were homogenized with 20 strokes of a motor-driven (2,500 rpm) Teflon pestle in a Potter homogenizer in a medium consisting of 0.25 M sucrose and 10 mM Tris at pH 7.4. The homogenate was centrifuged at 15,000 g for 15 min, and the supernatant was then collected and centrifuged again at 105,000 g for 1 h. The microsomal pellet obtained was resuspended in the homogenization medium and kept in liquid nitrogen until use. All of these procedures were carried out at 0-4°C.
Enzymatic assays. Glandular lactate dehydrogenase (EC 1.1.1.27) and phosphoglucose isomerase (EC 5.3.1.9) activities were measured by a standard spectrophotometric method (9). H+-K+-ATPase (EC 3.6.1.3) activity was assayed in gastric gland microsomes, basically according to the method reported by Hersey et al. (21). Thus gastric gland microsomes (0.1 mg of protein), previously incubated with or without different concentrations of ethanol (0.5-20% vol/vol) for 10 min at 37°C, were added to an assay mixture containing 150 mM KCl, 10 mM PIPES, 1 mM MgSO4, 5 mM MgATP, 10 µg/ml valinomycin, 10 µM DCCD, 2.5 µg/ml oligomycin, 1 mM EGTA, and 0.1 mM ouabain, pH 7.2, and the indicated ethanol concentrations. The reaction was carried out at 37°C for 20 min, and was stopped by adding one volume of 10% ice-cold trichloroacetic acid. After centrifugation (10,000 g for 2 min), inorganic phosphate was measured in the resulting supernatant (18). Nonspecific ATPase activity (measured under similar conditions but in the absence of valinomycin and substituting 250 mM sucrose for the 150 mM KCl in the reaction mixture) was subtracted from the estimated H+-K+-ATPase activity.
H+ transport assays. H+-K+-ATPase-dependent transport of protons across microsomal membranes was assayed using the fluorescent amine acridine orange as described elsewhere (23, 24). In this fluorometric assay, H+-K+-ATPase activity was coupled to H+ accumulation in microsomal vesicles via a K+/H+ exchange process. In response to the pH gradient generated by the H+-K+-ATPase activity, binding of acridine orange to intravesicular membrane sites increased, quenching the fluorescence of the probe. The assay was performed at room temperature (21-23°C) in a Perkin-Elmer LS-5B spectrofluorometer. Microsomes (40 µg of protein/ml) were suspended in a medium containing (in mM) 10 PIPES, 150 KCl, 1 MgCl2, 0.1 EDTA , and 1 ATP with 10 µM valinomycin, pH 7.2, in the absence or presence of different concentrations of ethanol (1-10% vol/vol). The excitation and emission wavelengths used were 493 and 530 nm, respectively. When the influence of ethanol on passive H+ permeability of microsomal membranes was studied, an ATP-dependent proton gradient across the microsomal membranes was generated first by the addition of 10 µM valinomycin. H+-K+-ATPase activity was then blocked by the rapid hydrolysis of ATP. This was achieved by the incorporation of glucose (5 mM) and hexokinase (18 U) into the reaction mixture immediately before the addition of either saline or ethanol (30).
ATP and cAMP assays. The ATP levels of gastric gland and parietal cells were determined fluorometrically according to the method reported by Lowry and Passonneau (25). Gastric gland suspensions or cultured parietal cells were incubated in the absence or presence of ethanol (1-20% vol/vol) at 37°C for 30 or 60 min; after addition of HClO4 (5%), the acid extracts were centrifuged and the supernatants were neutralized with imidazol-K2CO3 (0.3 M-3 M) and centrifuged again. ATP was measured in these neutralized extracts.
cAMP was determined in trichloroacetic acid extracts of cultured parietal cells using a radioimmunological method as previously described (15). Gastric parietal cells cultured for 24 h were incubated at 37°C for 15 min with different concentrations of ethanol in the absence or presence of either IBMX (0.5 mM) or forskolin (1 µM) or both. After treatment with 5% trichloroacetic acid and subsequent centrifugation (3.000 g for 15 min), cAMP was determined in the resulting supernatants.Statistical analysis. Statistical significance of differences was calculated by the paired Student's t-test. The differences were considered statistically significant when the P value was <0.05. The calculated concentrations of ethanol corresponding to the half-maximal effects (EC50) were calculated by the computer program Fig.P (Fig.P Software, Durham, NC).
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RESULTS |
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Effect of ethanol on trypan blue exclusion in gastric gland cells.
In the first part of the experiments, we studied the influence of
ethanol on the integrity of the cells present in the isolated rabbit
gastric glands. For this purpose, the percentage of cells stained with
trypan blue (0.1%) as well as the amount of both lactate dehydrogenase
and phosphoglucose isomerase released into the incubation medium were
estimated in gastric gland suspensions treated with different
concentrations of ethanol. As shown in Fig.
1, >98% of the cells excluded the
trypan blue stain in gastric glands incubated in the absence of
ethanol. The presence of ethanol (1-20% vol/vol) in the
incubation medium caused a time- and dose-dependent increase in the
percentage of stained cells. It should be pointed out that treatment of
gastric glands with 10% ethanol for 30 min was not associated with a
significant increase in the number of stained cells (1.0 ± 0.2%
and 3.5 ± 2.0% of stained cells, respectively, for gastric
glands incubated without and with 10% ethanol; P > 0.05; n = 12 experiments). However, higher
concentrations of ethanol (15 and 20%) significantly raised the amount
of stained cells, 19.3 ± 0.3% (n = 12 experiments) being the calculated EC50 value. Likewise,
after a longer period (60 min) of incubation, concentrations of ethanol
of 10% caused a marked increment in the percentage of stained cells
(92.1 ± 0.2% of stained cells for 10% ethanol); under these
conditions, the calculated EC50 value for ethanol was
8.2 ± 0.2%.
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Influence of ethanol on gastric acid secretion.
When the influence of different concentrations (1-20%) of ethanol
on the rate of acid formation was examined (Fig.
3), it was observed that 1% ethanol did
not significantly affect the rate of acid secretion in gastric glands
incubated either in the absence of secretagogues or in the presence of
forskolin (1 µM). However, 5% ethanol caused a significant reduction
in the rate of acid formation under both conditions (58% and 63%,
respectively). Maximal inhibition was already observed at 10% ethanol,
and the calculated EC50 values for this alcohol were
4.5 ± 0.2% and 3.5 ± 0.2%, respectively, in nonstimulated
and forskolin-stimulated gastric glands.
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Effect of ethanol on ATP and cAMP
levels in gastric glands and in cultured parietal cells.
As shown in Fig. 4, incubation of gastric
glands with 1% ethanol for either 30 or 60 min did not significantly
affect the concentration of ATP in isolated gastric glands. However,
concentrations of ethanol 5% caused a dose-dependent and
statistically significant decrease in ATP levels. It is of note that
treatment of gastric glands with 10% ethanol for 30 min reduced the
glandular concentration of ATP by 47% compared with that measured in
control gastric glands (2.0 ± 0.3 and 3.8 ± 0.2 nmol/mg dry
wt, respectively; P < 0.001, n = 3 experiments). The maximal effect (a reduction of ~80% in glandular
ATP content) was observed in the presence of 20% ethanol, with
8.8 ± 0.4% and 6.7 ± 0.1% being the corresponding
EC50 values estimated in gastric gland suspensions treated
for 30 and 60 min, respectively, with this alcohol.
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Effect of ethanol on
H+-K+-ATPase activity.
The influence of ethanol on H+-K+-ATPase
activity was studied in preparations of microsomes derived from
nonstimulated gastric glands. As indicated in MATERIALS AND
METHODS, microsomal H+-K+-ATPase activity
was assayed by a colorimetric method that measures the rate of
inorganic phosphate release from ATP (21). As shown in
Fig. 6,
H+-K+-ATPase activity was inhibited by the
presence of ethanol in the assay mixture in a dose-dependent manner.
The maximal inhibitory effect was observed at 20% ethanol, 8.5 ± 0.6% being the calculated EC50 value. It is of note that
at the lower ethanol concentrations tested (1 and 5%),
H+-K+-ATPase activity was not significantly
affected. This is in contrast with the finding that 5% ethanol caused
a marked reduction in the rate of both basal and forskolin-stimulated
acid formation (Fig. 3).
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Influence of ethanol on
H+-K+-ATPase-dependent proton
transport.
The influence of ethanol on the rate of
H+-K+-ATPase-dependent proton transport into
microsomal vesicles is shown in Fig. 7. Addition of ATP to the assay mixture caused a small and restricted reduction of acridine orange fluorescence (Fig. 7, inset),
possibly due to the presence of limited amounts of K+
within the microsomal vesicles. The additional presence of the K+ ionophore valinomycin increased the availability of
K+ to internal sites, maximally stimulating
H+-K+-ATPase activity. Consequently, the
ATP-induced intravesicular H+ accumulation rose, and the
acridine fluorescence decreased to a minimum value within a few
minutes, remaining at this low level for at least 15 min (data not
shown). This was caused by the continuous influx of protons mediated by
H+-K+-ATPase activity, which was apparently
equilibrated with the rate of spontaneous proton leakage from the
microsomal vesicles. The initial rate of this valinomycin-induced
decrease of fluorescence was considered an index of the rate of
H+-K+-ATPase-dependent proton transport into
microsomal vesicles (23, 24). The subsequent addition of
the K+/H+ exchange ionophore nigericin to the
assay mixture rapidly dissipated the pH gradient generated, and the
acridine fluorescence returned to its initial value in a few seconds
(Fig. 7, inset). As shown in Figs. 7 and
8, the presence of different
concentrations of ethanol in the reaction mixture caused a
dose-dependent reduction in the rate of
H+-K+-ATPase-dependent proton transport. The
maximal effect was already observed at 7.5% ethanol, 3% being the
calculated EC50 value.
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Effect of ethanol on passive H+
permeability.
To study more specifically the influence of ethanol on passive proton
permeability of microsomal membranes, we used the assay described by
Reichstein et al. (30). In this approach, an ATP-dependent H+ gradient across the microsomal membranes was generated
first by the addition of valinomycin (Fig.
9, inset). Afterwards,
H+-K+-ATPase activity was blocked by the rapid
hydrolysis of ATP through a glucose-hexokinase trap. In a few seconds,
spontaneous recovery of acridine orange fluorescence was observed,
which corresponded to the rate of passive H+ leakage from
the microsomal intravesicular space (30). In fact, when
the maximal fluorescence recovery was attained, nigericin was without
further effect (Fig. 9, inset). The presence of ethanol increased the rate of passive H+ leakage in a
dose-dependent manner, 7.9% (vol/vol) being the calculated
EC50 value (Figs. 9 and
10). As expected, nigericin (5 µM)
also accelerated the passive leakage of protons from the intravesicular
space (Fig. 9).
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DISCUSSION |
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It has been estimated that gastric intraluminal concentrations of
ethanol may attain values of up to 10% (vol/vol) even in moderate
drinkers (4, 25a). As demonstrated by Davenport (14), ethanol concentrations >8% (vol/vol) in the lumen of the stomach may
alter the gastric mucosal "barrier" in dogs. Furthermore, studies
carried out in animals (16, 22, 32, 34) and in humans
(33, 35) demonstrated that exposure of gastric mucosa to
concentrations of ethanol 10% produces functional and
histopathological changes, the extent of the injury being directly
related to the concentration of ethanol and to the exposure time. With
respect to the toxic effects of ethanol in gastric acid secretion,
concentrations of ethanol >10% have been shown to exert a marked
inhibitory effect (11, 30, 32, 33, 35) through a
multifocal mechanism acting at different points of the
stimulus-secretion coupling process (26, 30).
In this work, we have investigated the influence of different concentrations of ethanol (1-20% vol/vol) on the rate of acid formation in isolated rabbit gastric glands incubated either under basal conditions or in the presence of forskolin (1 µM). Thus we have observed that the presence of ethanol in the incubation medium for 30 min caused a dose-dependent reduction in the rate of acid formation in both nonstimulated and forskolin-stimulated gastric glands; the calculated EC50 values for ethanol were, respectively, 4.5 ± 0.2% and 3.5 ± 0.2%, values very similar to those obtained by Mazzeo et al. (26) (4.2% vol/vol) in their study of the inhibition by ethanol of the [14C]aminopyrine uptake ratio in basal parietal cell suspensions. Furthermore, the EC50 value for ethanol inhibiting [14C]aminopyrine uptake in forskolin-stimulated gastric glands, calculated from the data reported by Reichstein et al. (30), was also ~4%. In agreement with the latter authors, we have also found that a concentration of 10% ethanol was sufficient to cause maximal inhibition of acid formation in gastric glands incubated either under basal conditions or in the presence of forskolin. The fact that treatment of gastric glands with 10% ethanol for 30 min did not significantly modify the percentage of cells that excluded the trypan blue stain or the rates of lactate dehydrogenase and phosphoglucose isomerase release into the incubation medium indicates that the blockade in gastric acid formation elicited by this concentration of ethanol was not caused by a reduction in the viability of parietal cells present in the gastric glands.
Because gastric acid secretion is an energy-dependent process, a decrease in the cellular ATP levels caused by ethanol could be implicated in the underlying mechanism by which this alcohol inhibits the rate of acid formation in isolated gastric glands. In fact, ethanol caused a dose-dependent diminution in the ATP content of both gastric glands and parietal cells. However, the reduction in ATP levels did not appear to be the main mechanism involved in the inhibition of the rate of acid formation, at least by low concentrations of ethanol. This conclusion is based on the marked difference observed between the calculated EC50 value for ethanol-induced inhibition of acid formation in nonstimulated gastric glands (4.5 ± 0.2%) and those corresponding to ethanol-mediated reduction of ATP levels either in isolated gastric glands or in cultured parietal cells (8.8 ± 0.4% and 8.5 ± 0.2%, respectively).
The generation of cAMP has also been demonstrated to be a key step in
the modulation of gastric acid secretion by ethanol. Thus several
reports have established a direct relationship between the biphasic
effect of ethanol on acid formationstimulatory at concentrations of
ethanol <5% (vol/vol) and inhibitory at higher concentrations
and
the ability of this alcohol to elevate or depress the mucosal
(29) or glandular (30) content of cAMP. In
good agreement with these reports, our results show that ethanol, at concentrations of 5 and 10% (vol/vol), caused a significant reduction of cAMP levels in forskolin-stimulated parietal cells, whereas in cells
incubated under basal conditions the decrease in the glandular cAMP
content was statistically significant only in the presence of 10%
ethanol. However, our findings do not support the concept that
reduction in the glandular concentration of cAMP is the main mechanism
implicated in the blockade of acid secretion, not only in gastric
glands incubated under basal conditions but also in those treated with
forskolin. In fact, although 10% ethanol completely blocked acid
formation in forskolin-stimulated gastric glands and reduced the
content of cAMP in forskolin-treated parietal cells by ~95%, the
cellular concentration of this second messenger remained threefold
higher than that measured in parietal cells incubated under basal
conditions in the absence of ethanol (Table 1).
We also studied the influence of ethanol on H+-K+-ATPase activity in microsomes isolated from nonstimulated rabbit gastric glands. H+-K+-ATPase is located at the apical and tubulovesicular membranes of parietal cells and has been identified as the molecular machinery for proton transport. When this enzyme activity was assayed in terms of the hydrolysis of ATP according to the colorimetric method described above, we observed that low concentrations of ethanol (1 and 5%) did not significantly affect this activity. In contrast, higher ethanol concentrations (7.5-20%) caused a significant and dose-dependent inhibition of microsomal H+-K+-ATPase activity, 8.5 ± 0.6% being the calculated EC50 value for ethanol. Similar findings were reported in studies carried out in rabbit gastric glands (30) and parietal cells (26). In these reports, and in good agreement with our results, the calculated concentrations of ethanol corresponding to the half-maximal inhibitory effects (EC50 values) were ~8% and 10.5%, respectively. However, an apparent discordance arose from our data when the effects of ethanol on acid formation and on H+-K+-ATPase activity were compared. Thus 5% ethanol was able to reduce acid formation by ~60% in both nonstimulated and forskolin-stimulated gastric glands (Fig. 3), whereas H+-K+-ATPase activity was not significantly affected by this ethanol concentration (Fig. 6). This apparent inconsistency was reinforced by the difference found between the calculated EC50 values for ethanol as inhibitor of basal acid production and of H+-K+-ATPase activity (4.5 ± 0.2% and 8.5 ± 0.6%, respectively). These findings indicate that the inhibition of the hydrolytic activity of microsomal H+-K+-ATPase is less sensitive to ethanol than the reduction in acid formation by isolated gastric glands. Consequently, none of these results supports the concept that the inhibition of the hydrolytic activity of H+-K+-ATPase is the main mechanism involved in the inhibitory effect of ethanol on gastric acid production. It is of note that a similar discrepancy between the sensitivity to ethanol of aminopyrine uptake ratio and that of K+-stimulated para-nitrophenylphosphatase activity was reported by Mazzeo et al. (26) in isolated rabbit parietal cells (see Fig. 5 of Ref. 26).
When the influence of ethanol on the rate of H+-K+-ATPase-dependent proton transport into microsomal vesicles was studied, it was observed that the inhibition of this process caused by ethanol was more effective than that exerted by this alcohol on the hydrolytic activity of H+-K+-ATPase itself. In fact, 5% ethanol almost completely reduced (~90%) the estimated rate of H+-K+-ATPase-dependent proton transport (Fig. 8) without causing significant changes in the hydrolytic activity of this enzyme (Fig. 6). To explain this apparent discrepancy, we must consider that H+-K+-ATPase-dependent proton transport into microsomal membranes was estimated by measuring the initial rate of valinomycin-induced decrease of acridine orange fluorescence (see MATERIALS AND METHODS). With this in mind, the inhibition of this process elicited by low concentrations of ethanol that had no significant effect on the hydrolytic activity of the H+-K+-ATPase (e.g., 5% vol/vol) could be caused by 1) an increase in the passive proton permeability of microsomal membranes, which would dissipate the H+ gradient generated by the H+-K+-ATPase activity, and/or 2) an alteration of the H+-K+-ATPase itself that would specifically impair the ATP-dependent K+/H+ translocation process but preserve its hydrolytic activity. With respect to the first hypothesis, we have demonstrated, according to Reichstein et al. (30), that ethanol was able to accelerate the rate of passive H+ leakage from the microsomal intravesicular space in a dose-dependent manner. However, the difference found between the calculated EC50 values for ethanol increasing passive proton leakage (7.9% vol/vol) and inhibiting H+-K+-ATPase-dependent proton transport (3% vol/vol) excludes this first hypothesis. In fact, 5% ethanol almost completely blocked the H+-K+-ATPase-dependent proton transport (Fig. 8), with only small effects on passive proton leakage (Fig. 10). Concerning the second hypothesis, ethanol has a well-established "disordering" effect in cell membranes (13). Either high or low concentrations of ethanol may selectively disorder certain membrane lipid domains and thereby affect different functional proteins associated with these domains (26). In the case of gastric microsomal H+-K+-ATPase, the lipid environment has been shown to play a crucial role in maintaining its activity and stability. Thus changes in membrane-bound phospholipids elicited by lipid-perturbing agents such as ethanol (3, 26) or phospholipase A2 (27) markedly impair gastric H+-K+-ATPase activity. However, the mechanism by which low concentrations of ethanol may inhibit the H+-K+-ATPase-dependent transport of protons across microsomal membranes without affecting the hydrolytic activity of H+-K+-ATPase is not well understood. Nevertheless, it must be said that a similar phenomenon has been described in studies carried out in isolated gastric vesicles of hog stomachs, in which acetyl phosphate was used as substrate of the H+-K+-ATPase (1). These studies showed that acetyl phosphate was able to block the transport of protons in gastric vesicles whereas the hydrolytic activity of the H+-K+-ATPase was maintained. Furthermore, it is of note that the corresponding EC50 values calculated for ethanol as inhibitor of basal and forskolin-stimulated gastric acid secretion (4.5 ± 0.2% and 3.5 ± 0.2%, respectively) are much closer to those obtained for this alcohol in the assays of H+-K+-ATPase-dependent proton transport (3%) than in the passive proton permeability studies (7.9%). This suggests that, at least at low concentrations of ethanol (up to 5% vol/vol), the reduction in gastric acid secretion is mainly due to the inhibition of ATP-dependent K+/H+ translocation across the cell membranes and not to an increase of passive proton leakage, which may be relevant at higher concentrations of ethanol.
In conclusion, our results show that ethanol can inhibit gastric acid
secretion in isolated rabbit gastric glands by a multifocal mechanism,
affecting different cellular processes whose relative relevance in the
reduction of acid formation depends on their particular sensitivity to
ethanol toxicity. Thus high concentrations of ethanol (7% vol/vol)
exert a wide variety of toxic effects, including alteration of both
passive and H+-K+-ATPase-dependent transport of
protons across cell membranes, inhibition of the hydrolytic activity of
H+-K+-ATPase itself, reduction of the cellular
contents of both ATP and cAMP, and stimulation of the leakage of
cytosolic enzymes, which together may lead to a complete blockade of
acid secretion and even to a loss of parietal cell viability. However,
at lower concentrations of ethanol (up to 5% vol/vol), the inhibition
of H+-K+-ATPase-dependent translocation of
protons across the cell membranes seems to be the main mechanism
involved in decreasing gastric acid formation.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. J. C. Sánchez-Gutiérrez and Dr. J. J. Carrillo for their critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by a grant from Boehringer Ingelheim España S.A. J. C. Del Valle and R. Andrade are fellows of Boehringer Ingelheim España S.A., M. Salvatella is a fellow of Consejo Superior de Investigaciones Científicas, Spain.
Address for reprint requests and other correspondence: J. E. Felíu, Dept. de Bioquímica, Fac. de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4, 28029-Madrid, Spain (E-mail: juane.feliu{at}uam.es).
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 18 May 2000; accepted in final form 11 January 2001.
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REFERENCES |
---|
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---|
1.
Asano, S,
Kamiya S,
and
Takeguchi N.
The energy transduction mechanism is different among P-type ion-transporting ATPases.
J Biol Chem
267:
6560-6595,
1992.
2.
Aures, D,
Guth PH,
Paulsen G,
and
Grossman MI.
Effect of increased gastric mucosal histamine on alcohol-induced gastric damage in rats.
Dig Dis Sci
27:
347-352,
1982[ISI][Medline].
3.
Bailey, RE,
Nandi J,
Levine RA,
Ray TK,
Borer PN,
and
Levy GC.
NMR studies of pig gastric microsomal H+,K+-ATPase and phospholipids dynamics. Effects of ethanol perturbation.
J Biol Chem
261:
11086-11090,
1986
4.
Ballard, HI,
Wilkes JM,
and
Hirst BH.
Effect of alcohols on gastric and small intestinal apical membrane integrity and fluidity.
Gut
29:
1648-1653,
1988[Abstract].
5.
Beales, ILP,
and
Calam J.
Effect of N-methyl-histamine on acid secretion in isolated cultured rabbit parietal cells: implications for Helicobacter pylori associated gastritis and gastric physiology.
Gut
40:
14-19,
1997[Abstract].
6.
Becker, HD,
Reeder DD,
and
Thompson JC.
Gastrin release by ethanol in man and in dogs.
Ann Surg
179:
906-909,
1974[ISI][Medline].
7.
Berglindh, T,
Helander HF,
and
Öbrink KJ.
Effects of secretagogues on oxygen consumption, aminopyrine accumulation and morphology in isolated gastric glands.
Acta Physiol Scand
97:
401-414,
1976[ISI][Medline].
8.
Berglindh, T,
and
Öbrink KJ.
A method for preparing isolated glands from the rabbit mucosa.
Acta Physiol Scand
96:
150-159,
1976[ISI][Medline].
9.
Bergmeyer, HU.
Methods of Enzymatic Analysis. New York: Academic, 1974, p. 574-579, 1113-1117.
11.
Chacín, J,
Cárdenas P,
Lobo P,
and
Hernández I.
Secretory and metabolic effects of ethanol in the isolated amphibian gastric mucosa.
Gastroenterology
100:
1288-1295,
1991[ISI][Medline].
12.
Chew, CS,
Hersey SJ,
Sachs G,
and
Berglindh T.
Histamine responsiveness of isolated gastric glands.
Am J Physiol Gastrointest Liver Physiol
238:
G312-G320,
1980
13.
Chin, JH,
and
Goldstein DB.
Membrane disordering action of ethanol.
Mol Pharmacol
19:
425-431,
1981[Abstract].
14.
Davenport, HW.
Ethanol damage to canine oxyntic glandular mucosa.
Proc Soc Exp Biol Med
126:
657-662,
1967.
15.
Del Valle, JC,
Olea J,
Pereda C,
Gutiérrez Y,
and
Felíu JE.
Sulfonylurea effects on acid and pepsinogen secretion in isolated rabbit gastric glands.
Eur J Pharmacol
343:
225-232,
1998[ISI][Medline].
16.
Dinoso, VP,
Chuang J,
and
Murthy SN.
Changes in mucosal and venous histamine concentrations during instillation of ethanol in the canine stomach.
Dig Dis Sci
21:
93-97,
1976.
17.
Eysselein, VE,
Singer MV,
Wentz H,
and
Goebell H.
Action of ethanol on gastrin release in the dog.
Dig Dis Sci
29:
12-18,
1984[ISI][Medline].
18.
Fiske, CH,
and
Subbarow Y.
Colorimetric determination of phosphorus.
J Biol Chem
66:
375-400,
1925
20.
Harris, RA,
and
Simon FR.
Membrane fluidity and alcohol actions.
Curr Alcohol
8:
379-404,
1981[Medline].
21.
Hersey, SJ,
Steiner L,
Matheravidathu S,
and
Sachs G.
Gastric H+-K+-ATPase in situ: relation to secretory state.
Am J Physiol Gastrointest Liver Physiol
254:
G856-G863,
1988
22.
Kvietys, PR,
Twohig B,
Danzell J,
and
Specian R.
Ethanol-induced injury to the rat gastric mucosa.
Gastroenterology
98:
909-920,
1990[ISI][Medline].
23.
Lee, HC,
Breitbart H,
Berman M,
and
Forte JG.
Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles.
Biochim Biophys Acta
553:
107-131,
1979[ISI][Medline].
24.
Lee, HG,
and
Forte JG.
A study of H+ transport in gastric microsomal vesicles using fluorescent probes.
Biochim Biophys Acta
508:
339-356,
1978[ISI][Medline].
25.
Lowry, OH,
and
Passonneau JV.
A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p. 153-154.
25a.
Majchrowicz, E,
and
Noble EP.
Biochemistry and Pharmacology of Ethanol. New York: Plenum, 1979, p. 551-586.
26.
Mazzeo, AR,
Nandi J,
and
Levine RA.
Effects of ethanol on parietal cell membrane phospholipids and proton function.
Am J Physiol Gastrointest Liver Physiol
254:
G57-G64,
1988
27.
Nandi, J,
Wright MV,
and
Ray TK.
Effects of phospholipase A2 on gastric microsomal H+,K+-ATPase system: role of boundary lipids and the endogenous activator protein.
Biochemistry
22:
5814-5821,
1983[ISI][Medline].
28.
Oates, PJ,
and
Hakkinen J.
Studies on the mechanism of ethanol-induced gastric damage in rats.
Gastroenterology
94:
10-21,
1988[ISI][Medline].
29.
Puurunen, J,
Karppanen H,
Kairaluoma M,
and
Larmi T.
Effects of ethanol on the cyclic AMP system of the dog gastric mucosa.
Eur J Pharmacol
38:
275-279,
1976[ISI][Medline].
30.
Reichstein, BJ,
Okamoto C,
and
Forte JG.
Effect of ethanol on acid secretion by isolated gastric glands from rabbit.
Gastroenterology
91:
439-447,
1986[ISI][Medline].
31.
Rossi, I,
Olea J,
Herias M,
Pereda C,
and
Felíu JE.
Fructose 2,6-bisphosphate levels and modulation of glycolysis by histamine, cholecystokinin and forskolin in isolated rabbit gastric glands.
Metabolism
41:
339-344,
1992[ISI][Medline].
32.
Rutten, MJ,
and
Ito S.
Structural and functional changes by ethanol on in vitro guinea pig mucosa.
Am J Physiol Gastrointest Liver Physiol
251:
G518-G528,
1986[ISI][Medline].
33.
Singer, MV,
Leffmann C,
Eysselein WE,
Calden H,
and
Goebell H.
Action of ethanol and some alcoholic beverages on acid secretion and release of gastrin in humans.
Gastroenterology
93:
1247-1254,
1987[ISI][Medline].
34.
Tarnawski, A,
Hollander D,
and
Gergely H.
Ethanol-induced gastric mucosal injury: sequential analysis of morphologic and functional changes.
Gastroenterol Clin Biol
12:
88-92,
1985.
35.
Tarnawski, A,
Hollander D,
Stachura J,
Klimczyk B,
and
Bogdal J.
Alcohol injury to the normal human gastric mucosa: endoscopic, histologic and functional assessment.
Clin Invest Med
10:
259-263,
1987[ISI][Medline].