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Vol. 284, Issue 1, 37-42, 1998
Department of Applied Pharmacology, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto, Japan
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We found that roxatidine stimulates mucus secretion and synthesis by cultured rabbit gastric mucosal cells. In this study, we examined the roles of the extracellular Ca++ and calmodulin in these effects of roxatidine. Reduction of the extracellular Ca++ concentration decreased the roxatidine-induced increases in mucus secretion and synthesis by gastric mucosal cells. Roxatidine concentration-dependently promoted Ca++ influx and caused an increase in intracellular Ca++. After the addition of roxatidine, the increases in the secretion and synthesis reflected those in Ca++ influx and intracellular Ca++ concentration and then disappeared as Ca++ influx and intracellular Ca++ concentration returned to the control level. The roxatidine-stimulated Ca++ influx and intracellular Ca++ mobilization were abolished by reduction of the extracellular Ca++ concentration. Nifedipine and diltiazem inhibited both the effects of roxatidine, but even at 10 µM, the inhibition was partial. Furthermore, W-7 (a calmodulin antagonist) completely abolished the effects of roxatidine on mucus secretion and synthesis without causing a reduction of the stimulated Ca++ influx. Taken together, these results suggest that roxatidine promotes Ca++ influx through both voltage-sensitive Ca++ channels and other Ca++ entry gates and the subsequent intracellular Ca++ mobilization, leading to potentiation of mucus secretion and synthesis by rabbit gastric mucosal cells. In addition, Ca++-activated calmodulin may play a pivotal role in these stimulatory effects of roxatidine.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Roxatidine
(roxatidine acetate hydrochloride, a histamine H2 receptor
antagonist) is known to exert a mucosal protective effect as well as an
antisecretory effect in rats (Tarutani et al., 1985a
; Tarutani et al., 1985b; Shiratsuchi et al., 1988;
Okabe et al., 1989; Naito et al., 1995). It has
been thought that the protective effect of roxatidine is due to
potentiation of defensive factors, including gastric mucus, and to
inhibition of aggressive factors such as acid and reactive oxygen
species. Ichikawa et al. (1994) reported that roxatidine
stimulates mucin biosynthesis by isolated rat stomachs. We also
reported that it directly acts on rabbit gastric mucosal cells,
inducing increases in both mucus secretion and synthesis (Takahashi and
Okabe, 1995). Such stimulatory effects were observed with roxatidine,
but not with other H2 antagonists such as cimetidine,
ranitidine and famotidine, which indicates that the blocking of
H2 receptors alone does not stimulate mucus secretion. In
addition, our previous study revealed that neither prostaglandins nor
nitric oxide are involved in the action of roxatidine. In the present
study, we investigated the roles of extracellular Ca++ and
calmodulin in the roxatidine-stimulated mucus secretion and synthesis
by gastric mucosal cells.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Preparation of gastric mucosal cells.
Gastric mucosal cells
were prepared from rabbit stomachs according to the method of Watanabe
et al. (1994). Briefly, male Japanese White rabbits (Nihon S.L.C.,
Shizuoka, Japan), weighing 2.5-3.5 kg, were anesthesized with Nembutal
(50 mg/kg, i.v.; Abbott, North Chicago, IL). After a stomach had been
excised, the surface of the oxyntic mucosa was removed with a razor
blade and minced immediately. The minced tissue was incubated in
Hank's balanced salt solution containing 0.07% collagenase (Wako
Chemicals, Osaka, Japan) for 15 min at 37°C, and then washed with
Ca2+, Mg2+-free Hank's solution containing 1 mM EDTA and 1 mg/ml bovine serum albumin. These procedures were
repeated twice. The mucosal cells were obtained by filtration through
metal meshes (diameters, 300 µm and 100 µm). The viability of the
isolated cells was more than 85%, as determined by the dye exclusion
test (Phillips, 1973).
Cell culture.
Coon's modified Ham's F12 medium (GIBCO BRL,
Gaithersburg, MD) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin and 0.25 µg/ml
amphotericin B, and culture plates and dishes coated with collagen type
I (Sigma Chemicals, St. Louis, MO) were used. The medium contained 2 mM
CaCl2. Gastric mucosal cells (2 × 105
cells/1 ml) were inoculated onto 12-well plates (Corning & Costar, Corning, NY). For measurement of intracellular Ca2+
concentration, cells were inoculated onto glass-bottomed 35 mm-dishes (Mat Tek, Ashland, MA). The cultures were maintained at 37°C under 5% CO2 in air, the medium being changed every day. The
cells reached confluence 2-3 days later. Most of the cultured cells
were morphologically epithelial-like, and 80-90% of them were
confirmed to be mucus-producing ones by periodic acid-Schiff staining,
as described previously (Takahashi and Okabe, 1995; Takahashi et al.,
1995; Takahashi and Okabe, 1996a).
Determination of mucus secretion and synthesis.
The amounts
of secreted and synthesized mucus were determined according to the
methods of Terano et al. (1982) and Keates and Hanson (1990)
with a slight modification. Gastric mucosal cells grown to confluence
were washed with PBS and then incubated with 1 ml of the medium
containing [3H] glucosamine (18.5 kBq, 1800 GBq/mmol; New
England Nuclear, Boston, MA) in the presence of the indicated drugs or
vehicle at 37°C. Unless otherwise stated, the Ca++
concentration in the medium was 2 mM. At appropriate times, the medium
was recovered and centrifuged at 3000 × g for 3 min,
and then an aliquot (0.8 ml) of the resulting supernatant was mixed with 0.2 ml of 50% trichloroacetic acid. The mixture was held on ice
for 5 min and then centrifuged at 10,000 × g for 5 min at 4°C. After the resulting pellet was solubilized with 0.25 ml of
5% Triton X-100, an aliquot (0.2 ml) was subjected to Sepharose CL-4B
column (4 ml) chromatography. The radioactivity in the void fractions
was measured as the amount of mucus secreted by the cells into the
medium. For estimation of mucus synthesis in the cells, the amount of
[3H] glucosamine incorporated into the cells was
determined. The remaining cells were washed twice with PBS and then
solubilized with 0.25 ml of 5% Triton X-100. As described above, when
an aliquot (0.2 ml) was loaded onto the same column, the radioactivity
in the void fractions was measured. About 80% of the precipitated labeled materials in the medium and cells were identified as mucus glycoprotein on gel filtration chromatography.
Determination of Ca++ influx into gastric mucosal
cells.
Ca++ influx was evaluated as
45Ca++ uptake by gastric mucosal cells.
45Ca++ uptake was determined according to the
method of Tanaka et al. (1990) with a slight modification.
After mucosal cells grown to confluence had been washed with PBS, they
were incubated in 0.5 ml of the medium containing 2 mM
45CaCl2 (22.2 kBq, >370 GBq/mmol; New England
Nuclear) at 37°C. At appropriate times, the cells were washed with
PBS and then solubilized with 0.2 ml of 0.3N NaOH. Then the
radioactivity in the lysate was measured.
Determination of intracellular Ca++ concentration. Intracellular Ca++ concentration was measured in the cells loaded with Fura-2. Gastric mucosal cells were incubated with 2 µM Fura-2AM (Dojindo Laboratories, Kumamoto, Japan) for 1 hr at 37°C. The cells were washed with medium and then held for 30 min. After being washed with medium, the cells were incubated with 10 µM roxatidine or vehicle for 4 hr. Relative fluorescence intensity was monitored at an excitation wavelength of 340 nm/380 nm and an emission wavelength of 510 nm, with a digital fluorescence analyzer (Attofluor; Carl Zeiss, Obernkochen, Germany). Intracellular Ca++ concentration was calculated from the fluorescence intensity using an application software (Atto Graph).
Determination of cell viability.
Cell viability was
determined by the mitochondrial function and membrane permeability
assays, as described previously (Takahashi and Okabe, 1996b).
Mitochondrial function was assessed by the colorimetric method
involving MTT (Sigma Chemicals). After mucosal cells had been incubated
with 10 µM roxatidine or vehicle for 4 hr, 0.2 ml of 5 mg/ml MTT
solution was added. Two hours later, the MTT was extracted with 3 ml of
isopropanol containing 0.04 N HCl, and the color change of the extract
was measured at 595 nm. The membrane permeability was assessed by the
dye exclusion method. Cells were washed after incubation with 10 µM
roxatidine or vehicle, and then 0.2 ml of 0.1% trypan blue solution
was added. Three minutes later, the numbers of stained and nonstained
cells were determined in four randomly chosen fields in each well under a microscope (CK2; Olympus, Tokyo, Japan; ×100). Cell viability was
determined as Nonstained cells/(Stained cells + Nonstained cells).
Drugs. Roxatidine (Teikoku Hormone Mfg. Co., Tokyo, Japan), nifedipine (Sigma Chemicals), diltiazem (Tanabe Seiyaku Co., Osaka, Japan) and W-7 (Seikagaku Corp., Tokyo, Japan) were dissolved in dimethyl sulfoxide. For each assay, dimethyl sulfoxide was diluted to a final concentration of less than 0.8% in the medium. All other chemicals were of reagent grade.
Statistical analysis. Data are presented as means ± S.E. Statistical differences were evaluated using Student's t -test or Dunnett's multiple comparison test, and a P value < .05 was regarded as significant.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of extracellular Ca++ concentration on roxatidine-induced increases in mucus secretion and synthesis. We examined the effects of extracellular Ca++ concentration on the roxatidine-induced increases in mucus secretion and synthesis by gastric mucosal cells (fig. 1). At 2 mM extracellular Ca++, exposure to 10 µM roxatidine for 4 hr induced significant increases in mucus secretion and synthesis. However, reduction of the extracellular Ca++ concentration caused decreases in the roxatidine-stimulated secretion and synthesis in a concentration-dependent manner, and reduction to 0.002 mM completely abolished these stimulatory effects of roxatidine. Basal secretion and synthesis also decreased with reduction of the extracellular Ca++ concentration, but the cells apparently secreted and synthesized mucus even at 0.002 mM extracellular Ca++. At 0.02 mM extracellular Ca++, the basal secretion and synthesis were not different from the corresponding secretion and synthesis in the presence of 2 mM extracellular Ca++. In contrast, the roxatidine-stimulated secretion and synthesis were significantly reduced compared with those at 2 mM extracellular Ca++. As determined by both MTT and dye exclusion methods, cell viability was similar among cells incubated at 2 mM and 0.002 mM extracellular Ca++ in the presence and absence of roxatidine for 4 hr.
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Stimulatory effect of roxatidine on Ca++ influx into gastric mucosal cells. We examined whether roxatidine promotes Ca++ influx into gastric mucosal cells (fig. 2). Mucosal cells incorporated extracellular Ca++ without any external stimulus for 2 hr. Roxatidine caused a concentration-dependent increase in Ca++ influx, and significant effects were observed at 1 and 10 µM, the increases being 14.2% and 21.8%, respectively.
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Effects of nifedipine and diltiazem on roxatidine-induced increases in Ca++ influx, mucus secretion and synthesis. To determine whether voltage-sensitive Ca++ channels are involved in the roxatidine action on Ca++ mobilization, we examined the effects of two Ca++ channel blockers, nifedipine and diltiazem, on the stimulated Ca++ influx (fig. 5A). Basal Ca++ influx for 2 hr was not affected by nifedipine or diltiazem, even at 10 µM. The roxatidine (10 µM)-induced increase in Ca++ influx was slight (around 12%) but was significant even in the presence of nifedipine and diltiazem, as compared with that in the corresponding cells without roxatidine. However, nifedipine and diltiazem significantly reduced the roxatidine-stimulated Ca++ influx by 60.8% and 66.3%, respectively. Similarly, the increase (10-15%) in intracellular Ca++ induced by 10 µM roxatidine at any time-point was also observed, but it was reduced by nifedipine and diltiazem. As shown in figure 5B, the inhibition at 2 hr by 10 µM nifedipine was 52.4% and by 10 µM diltiazem was 56.0%
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Effects of W-7 on roxatidine-induced increases in Ca++ influx, mucus secretion and synthesis. To evaluate further the roles of intracellular Ca++ in mucus secretion and synthesis in response to roxatidine, we investigated the relation between Ca++ and calmodulin. The significant increase in Ca++ influx caused by 10 µM roxatidine was similarly observed even in the presence of 3 µM W-7 (a calmodulin antagonist). However, W-7 concentration-dependently inhibited the increases in both mucus secretion and synthesis caused by 10 µM roxatidine (fig. 7). At 3 µM, W-7 completely abolished the stimulatory effects of roxatidine on the secretion and synthesis of mucus.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We found that the stimulatory effects of roxatidine on mucus
secretion and synthesis by gastric mucosal cells are dependent on the
presence of extracellular Ca++. This suggests that
Ca++ influx plays an important role in the actions of
roxatidine. The important role of the extracellular Ca++ is
also evidenced by the following results. First, roxatidine concentration-dependently promoted Ca++ influx into cells,
and the concentration for the effect on Ca++ influx was
consistent with those for mucus secretion and synthesis (Takahashi and
Okabe, 1995). In addition, roxatidine caused an increase in
intracellular Ca++, but reduction of extracellular
Ca++ concentration abolished this response to roxatidine.
Second, roxatidine increased Ca++ influx and intracellular
Ca++ concentration, followed by enhancement of mucus
secretion and synthesis. The reductions of the increases in
Ca++ influx and intracellular Ca++ might lead
to disappearance of the stimulatory effects of roxatidine on mucus
secretion. Third, Ca++ channel blockers inhibited the
roxatidine-stimulated Ca++ influx, intracellular
Ca++ mobilization and mucus secretion and synthesis. Taken
together, these results indicate that the increased influx of
Ca++ caused by roxatidine might contribute to the increases
in mucus secretion and synthesis by gastric mucosal cells. Certainly,
reduction of the extracellular Ca++ concentration may
decrease the viability of cells, resulting in the loss of a cell
response to roxatidine, because reduction in extracellular
Ca++ was accompanied by decreases in basal secretion and
synthesis of mucus. We confirmed that complete depletion of
extracellular Ca++ for 4 hr by EGTA induces cell damage.
However, the above possibility is unlikely, because the cell viability
was not affected by reduction of the extracellular Ca++
concentration or by treatment with roxatidine. In addition, reduction of the extracellular Ca++ concentration to 0.002 mM
significantly inhibited the roxatidine-stimulated secretion and
synthesis of mucus, although the reduction slightly affected basal
secretion and synthesis.
Previous studies revealed that extracellular Ca++-dependent
and -independent pathways of gastric mucus secretion exist (Seidler and
Sewing, 1989; Micots et al., 1993; Hata et al.,
1994). In the case of roxatidine, it is apparent that the drug
potentiates extracellular Ca++-dependent secretion of
mucus. It is of note that the inhibition by nifedipine and diltiazem of
the roxatidine-induced increases in Ca++ influx,
intracellular Ca++ and mucus secretion and synthesis was
not complete even at 10 µM, although such channel blockers have been
widely used for complete blocking of voltage-sensitive Ca++
channels at around 1 µM in cell culture. Furthermore, reduction of
extracellular Ca++ concentration abolished the stimulatory
effect of roxatidine on intracellular Ca++ mobilization.
Our results also indicate that roxatidine induces the increase in
Ca++ influx through both voltage-sensitive Ca++
channels and other Ca++ entry gates, resulting in elevation
of intracellular Ca++ concentration. As reported by
Tepperman et al. (1991), rabbit gastric mucosal cells may
possess voltage-sensitive Ca++ channels. However, it is
unlikely that voltage-sensitive Ca++ channels are involved
in the basal secretion and synthesis of mucus, because nifedipine and
diltiazem did not affect basal Ca++ influx, intracellular
Ca++ concentration or mucus secretion and synthesis.
Tepperman et al. (1991) also stated that under physiological
conditions, it is uncertain whether voltage-sensitive Ca++
channels contribute to the regulation of gastric mucosal cell functions. Considering that the extracellular Ca++
concentration influenced basal mucus secretion and synthesis, it seems
that other Ca++ gates play crucial roles in basal secretion
as well as in the roxatidine-stimulated secretion. In fact,
Ca++ influx into gastric mucosal cells constitutively
occurred in the presence and absence of Ca++ channel
blockers. Such Ca++ entry gates remain unidentified, so
further investigation is needed.
Given the finding that W-7 potently inhibited the roxatidine-induced increases in mucus secretion and synthesis, calmodulin might play a pivotal role in the actions of roxatidine. It is well established that calmodulin is activated by Ca++ -binding, and Ca++/calmodulin subsequently exerts numerous biological effects in cells. Accordingly, calmodulin might mediate the action of the Ca++ influx induced by roxatidine in gastric mucosal cells. In addition, calmodulin may be involved in basal secretion and synthesis of mucus, because basal secretion and synthesis were significantly reduced by W-7 at high concentrations.
It has been reported that sustained Ca++ overloading of
gastric mucosal cells causes cell injury (Tepperman et al.,
1991; Tepperman and Soper, 1993). In the case of roxatidine treatment,
it is apparent that the roxatidine-induced increase in the
intracellular Ca++ does not induce cell injury, because
cell viability was not affected by treatment with roxatidine. In fact,
roxatidine caused a slow increase in intracellular Ca++ for
about 2 hr, but the degree of the increase was lower (about 20-30 nM),
which indicates that roxatidine does not induce Ca++
overloading. In contrast, when Ca++ overloading was induced
by treatment of cells with 1 µM ionomycin, Ca++ influx
and intracellular Ca++ mobilization were markedly enhanced
to about 5-fold of the basal level and were sustained thereafter. In
gastric mucosal cells, the basal concentration of intracellular
Ca++ was higher (about 100 nM) than those in other cells.
Tepperman and Soper (1993) obtained a similar result. These results
suggest that gastric mucosal cells may intrinsically possess resistance to toxicity because of slightly higher intracellular Ca++
concentration. Moreover, the elevated Ca++ influx,
intracellular Ca++ concentration and mucus secretion and
synthesis induced by roxatidine returned to control levels, which
suggests some regulation of responses to roxatidine. However, although
the elevated intracellular Ca++ concentration caused by
roxatidine returned to the control level within 3 hr, the stimulation
of Ca++ influx persisted longer. This suggests that the
Ca++ that results from the influx might be rapidly utilized
by intracellular molecules, including calmodulin, and that a
Ca++ extrusion mechanism might be activated. Elucidation of
these regulation mechanisms awaits further evaluation.
Recently, Ciacci et al. (1996) reported that ranitidine and
roxatidine stimulate the proliferation of human gastric cancer cells in
culture. They found that stimulation of MKN28 cell proliferation was
observed over 12 hr after the addition of such H2 receptor antagonists. It is apparent that the enhancement of mucus secretion and
synthesis does not result from an increase in the cell number, because
the effects of roxatidine on mucus secretion and synthesis became
maximum at 4 hr and had disappeared at 12 hr.
The concentration of roxatidine used in the present study (1-10 µM)
is considered comparable to therapeutic dose in vivo. Lameire et al. (1988) reported that p.o. administration of
roxatidine at 150 mg to humans gives an intragastric concentration of
about 4 mM and a plasma concentration of around 1 µM for 6 hr. In
addition, a concentration of more than 10 µM is required for 80%
inhibition of histamine-stimulated [14C]aminopyrine
accumulation by isolated rabbit gastric glands (Bickel et
al., 1988).
The target site of roxatidine for stimulation of mucus secretion and
synthesis remains unclear. We previously reported that the stimulatory
effect of roxatidine might not be mediated through H2
receptors (Takahashi and Okabe, 1995). In preliminary experiments using
a transwell chamber, we found that roxatidine from both the apical and
the basolateral sides can enhance mucus synthesis (data not shown). It
is known that gastric epithelial cells show cell polarity, and membrane
proteins are specifically localized on either the apical membrane or
the basolateral membrane, depending on their properties. These
observations suggest that the target site may exist inside, not on the
membranes. Further investigation is ongoing.
Taken together, these results suggest that roxatidine promotes Ca++ influx through both voltage-sensitive Ca++ channels and other Ca++ entry gates and the subsequent intracellular Ca++ mobilization, leading to potentiation of mucus secretion and synthesis by rabbit gastric mucosal cells. In addition, calmodulin, activated by inwardly moving Ca++, may play a pivotal role in these stimulatory effects of roxatidine.
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Acknowledgments |
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We wish to thank Mr. N. J. Halewood for his critical reading of the manuscript and Mr. T. Tsukahara for his technical assistance.
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Footnotes |
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Accepted for publication September 30, 1997.
Received for publication April 28, 1997.
1 This research was supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan (Grant-in-Aid for Encouragement of Young Scientists #08772122).
Send reprint requests to: Satoru Takahashi, Ph.D., Department of Applied Pharmacology, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan.
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Abbreviations |
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PBS, Dulbecco's modified Ca++, Mg++-free phosphate-buffered saline; MTT, 3-(4, 5-dimethyl-2-thiazoyl)-2, 5-diphenyl-2H-tetrazolium bromide; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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indicate statistically significant differences from
the corresponding vehicle-treated cells, roxatidine-treated cells at 2 mM extracellular Ca++ and vehicle-treated cells at 2 mM
extracellular Ca++, respectively.
