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1 Department of Applied
Pharmacology, It has been reported that cyclooxygenase-2
(COX-2) may play a crucial role in gastric ulcer healing. We examined
the localization of COX-2 and the regulation of COX-2 mRNA expression
in acetic acid ulcers in rats.
PGE2 production was elevated in
ulcerated tissue but not in intact tissue. COX-2 mRNA
expression was induced in only the ulcerated tissue, and COX-2 protein
was found in fibroblasts, monocytes/macrophages, and granulocytes. A
selective COX-2 inhibitor inhibited increased
PGE2 production by the ulcerated
tissue. Interleukin-1
prostaglandin; interleukin-1 IT IS WELL ESTABLISHED THAT PGs play a physiological
role in maintaining the integrity of the gastric mucosa. Nonsteroidal anti-inflammatory drugs (NSAIDs) induce gastric mucosal injury in rats
and humans (1, 28). Exogenous PGs protect the gastric mucosa against
various types of damage caused by necrotizers, including NSAIDs (17,
19). In addition, PGs are known to play an important role in the
healing of gastric ulcers. PG production is elevated in the ulcerated
gastric tissue in rats (23, 29), and treatment with NSAIDs causes a
delay of ulcer healing in rats and humans (11, 13, 23, 29).
NSAIDs act on cyclooxygenase (COX), the key enzyme in PG formation
(27). COX exists in two isoforms, of which COX-1 is expressed in many
tissues, including the stomach, and COX-2 is induced in fibroblasts,
monocytes/macrophages, and other cell types at inflammatory sites (6,
15). The expression of COX-2 is stimulated by lipopolysaccharide, several growth factors, and cytokines such as interleukin-1 (IL-1) and
tumor necrosis factor- Therefore, to fully understand the role of COX-2 in ulcer healing, we
investigated the localization of COX-2 and the regulation of COX-2 mRNA
expression in acetic acid ulcers in rats.
Production of gastric ulcers.
Male Donryu rats (Nihon SLC, Hamamatsu, Japan), weighing 250-300
g, were fasted for 5 h before ulcer production. Under ether anesthesia,
gastric ulcers were induced by submucosal injection of 20% acetic acid
(0.04 ml) into the border between the antrum and the fundus on the
anterior wall of the stomach (24). After closure of the abdomen, the
rats were maintained in the usual manner. Because deep, well-defined
ulcers were observed 5 days after the acid injection, we defined the
fifth day as the day of ulceration
(day
0). At the indicated times, rats
were killed and their stomachs were excised. Subsequently, the stomachs
were incised along the greater curvature, and the ulcerated area
(mm2) was determined under a
dissecting microscope (magnification, ×10).
Determination of PGE2 production by
gastric tissue.
PGE2 production was assayed
according to the method of Lee and Feldman (12). Gastric specimens were
taken from both intact (posterior side) and ulcerated tissue (anterior
side) of stomachs with ulcers and from normal stomachs. The gastric
specimens were placed in 50 mM Tris · HCl (pH 8.4)
buffer and then minced with scissors. After the tissues were washed and
then resuspended in 1 ml of buffer, they were subjected to vortex
mixing at room temperature for 1 min to stimulate
PGE2 production, followed by
centrifugation at 10,000 g for
15 s. On stimulation with vortexing,
PGE2 production increased by
~2.5-fold, compared with the production without vortexing, in all
tissue examined. To examine the effects of COX inhibitors, we
preincubated tissue with the indicated concentrations of indomethacin, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide
(NS-398), or vehicle on ice for 10 min before stimulation. The amounts
of PGE2 in the resulting
supernatants were determined by enzyme immunoassay (PGE2 EIA kit; Cayman Chemical,
Ann Arbor, MI). PGE2 production was expressed as picograms of PGE2
per milligram of tissue per minute.
Preparation of 32P-labeled cDNA probes.
Rat COX-1 and COX-2 cDNA probes were prepared as described previously
(10). cDNA probes of rat IL-1 Northern blot analysis.
Gastric specimens were taken from both intact (posterior side) and
ulcerated tissue (anterior side) of stomachs with ulcers and from
stomachs without ulcers. Total RNAs were extracted from the specimens
by means of the acid-guanidinium thiocyanate-phenol-chloroform method
(2), using TRIzol (GIBCO BRL, Gaithersburg, MD).
Poly(A)+ RNAs were purified with
Oligotex(dT)30 (TaKaRa, Kyoto,
Japan). Poly(A)+ RNAs
(0.1-0.5 µg) were separated by electrophoresis on 1.2% agarose gels, transferred onto nylon membranes (Gene Screen Plus; NEN, Boston,
MA), and then hybridized with
32P-labeled cDNA probes (20). The
detection and quantification of hybridized mRNAs were carried out with
an imaging analyzer (BAS-5000Mac; Fuji Film, Tokyo, Japan). Unless
otherwise stated, the levels of the mRNAs were expressed as a ratio
against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Western blot analysis.
Gastric specimens were taken from both intact (posterior side) and
ulcerated tissue (anterior side) of stomachs with ulcers and from
stomachs without ulcers. COX proteins were partially purified from the
specimens as described by Gierse et al. (4). After aliquots (60 µg)
of the proteins eluted from a DEAE-Sepharose column and standard COX
proteins (0.1 µg; Cayman Chemical) had been subjected to SDS-PAGE
(10%), the separated proteins were electrophoretically transferred
onto Immobilon-P membranes (Millipore, Bedford, MA) as described by
Towbin et al. (26). The membranes were incubated with the antibody
against COX-1 or COX-2 protein after nonspecific binding sites had been
blocked. COX proteins were visualized by the avidin-biotin-peroxidase
complex (ABC) method, using a Vectastain ABC kit (Vector Laboratories,
Burlingame, CA) and 3,3'-diaminobenzidine tetrahydrochloride
(Dojindo Laboratories, Kumamoto, Japan).
Immunohistochemical analysis.
Gastric specimens were taken from both intact (posterior side) and
ulcerated tissue (anterior side) of stomachs with ulcers and from
stomachs without ulcers. After they had been fixed with 4%
paraformaldehyde in PBS, frozen sections (16 µm thick) were prepared.
The sections were incubated with anti-COX-2 antibody after deactivation
of endogenous peroxidase with 0.3%
H2O2
and blockage of nonspecific binding sites. COX-2 protein was visualized by the ABC method as described above. The sections were successively stained with hematoxylin.
Tissue culture.
On days
3 and
10, the ulcer base was excised and
divided into six fragments. The fragments were placed in the wells of
48-well culture plates (Corning & Costar, Corning, NY) and then
incubated in 0.5 ml of DMEM (GIBCO BRL) supplemented with 0.5% fetal
bovine serum (GIBCO BRL), 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 µg/ml amphotericin B in the presence of the indicated agents
at 37°C under 5% CO2 in air.
Forty-eight hours later, poly(A)+
RNAs were isolated from the fragments and subjected to Northern blot
analysis. In addition, PGE2
production assay was performed as described above. After a 48-h
incubation with the reagents, the contents (µg/g tissue) of DNA,
total RNA, and poly(A)+ RNA were
nearly similar, compared with those before culturing. Histologically,
granulation tissue remained unchanged, and fibroblasts, epithelial
cells, and immune cells were observed after the incubation.
Materials.
NS-398 and FR-167653 were kindly supplied by Taisho Pharmaceutical
(Tokyo, Japan) and Fujisawa Pharmaceutical (Osaka, Japan), respectively. Polyclonal rabbit antisera against ovine
COX-1 (PGH synthase 1) and mouse COX-2 (PGH synthase 2) were purchased
from Cayman Chemical. These antibodies have been known to cross-react with rat enzymes (8). Other agents and their sources were as follows:
indomethacin (Sigma Chemical, St. Louis, MO); synthesized oligonucleotides, Moloney murine leukemia virus RT, and
Taq DNA polymerase (GIBCO BRL); human
GAPDH cDNA probe (Clontech, Palo Alto, CA);
[ Statistical analysis.
Data are presented as means ± SE. Statistical differences in the
dose-response studies were evaluated by Dunnett's multiple comparison
test. Student's t-test was also
applied to comparisons between two groups.
P < 0.05 was regarded as
significant.
PGE2 production by gastric tissue with
ulcers.
On day
0, there were round, well-defined
ulcers in all animals, with the ulcerated area being 41.0 ± 4.8 mm2 (Fig.
1A). The ulcers
spontaneously healed. The areas decreased to 14.9 ± 2.4 and 4.9 ± 1.3 mm2 on
days
7 and
14, respectively.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
(IL-1), tumor necrosis factor-
(TNF-), and transforming growth factor-1 (TGF-1) mRNAs were
also expressed only in the ulcerated tissue. In a culture of isolated
ulcer base, blockade of IL-1 and TNF- reduced COX-2 mRNA
expression and PGE2 production. In contrast, COX-2 mRNA expression and
PGE2 production were promoted by
prevention of TGF-1 action. These results indicate that COX-2 protein is highly localized in the base of gastric ulcers in rats and
that COX-2 mRNA expression might be regulated positively by IL-1 and
TNF- and negatively by TGF-1.
; tumor necrosis factor-; transforming growth factor-1
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
(TNF-). A recent study by Mizuno et al.
(16) showed that increased PG production in gastric ulcers might result
from the induction of COX-2 and selective inhibition of COX-2 activity
may lead to impairment of ulcer healing in mice. However, the molecules
involved in COX-2 expression in gastric ulcers remain unclear.
METHODS
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Abstract
Introduction
Methods
Results
Discussion
References
, rat TNF-, and rat TGF-1 were
obtained by means of RT-PCR. Total RNAs for amplification of these
cDNAs were isolated from the spleen of a
lipopolysaccharide-infused rat for IL-1 and TNF- and the
liver of a 30%-hepatectomized rat for TGF-1. The primers used were
as follows: 5'-GAAGCTGTGGCAGCTACCTATGTCT-3' and
5'-CTCTGCTTGAGAGGTGCTGATGTAC-3' for IL-1,
5'-CACGCTCTTCTGTCTACTGA-3' and
5'-GGACTCCGTGATGTCTAAGT-3' for TNF-, and
5'-GCCTCCGCATCCCACCTTTG-3' and
5'-CGGGTGACTTCTTTGGCGT-3' for TGF-1. The products
corresponding to COX-1 (1,435 bp), COX-2 (1,484 bp), IL-1 (520 bp), TNF- (546 bp), and TGF-1 (396 bp) were purified from
polyacrylamide gels and used as probes after their sequences had been
confirmed to be completely identical to known ones (with reference to
the databases of GenBank and European Molecular Biology Laboratories).
The cDNA probes were 32P-labeled
by the random primer method (Ready-To-Go; Pharmacia Biotec, Uppsala,
Sweden).
-32P]dCTP (NEN);
IL-1 receptor antagonist (Pepro Tech, Rocky Hill, NJ); and anti-mouse
TNF- antibody, anti-human TGF-1 antibody, and anti-human TGF-
receptor II antibody, which react with rat TNF-, rat TGF-1, and
rat TGF- receptor II, respectively (Santa Cruz Biotechnology, Santa
Cruz, CA). All other chemicals used were of reagent
grade.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fig. 1.
Healing process of acetic acid-induced gastric ulcers and
PGE2 production by gastric tissue
of rats. At indicated times after gastric ulceration, the ulcerated
area (A) and
PGE2 production by intact and
ulcerated tissue (B) were
determined. PGE2 production by a
normal stomach without ulcers was also determined. Data are presented
as means ± SE (n = 6-8).
* Significantly different (P < 0.05) from normal tissue.
1 · min1
(Fig. 1B).
PGE2 production in the ulcerated
tissue significantly increased by about threefold during
days
0-7,
compared with that in the normal tissue. Thereafter, the production
decreased and returned to the normal level on
day
14. However,
PGE2 production in the intact
tissue was not affected by the presence of ulcers in the stomach. The
level was nearly the same as that in the normal tissue.
In addition, we examined the effects of COX inhibitors on
PGE2 production (Fig.
2). Indomethacin dose dependently inhibited PGE2 production by gastric tissue
with ulcers as well as that by normal gastric tissue. In contrast,
NS-398, a selective COX-2 inhibitor (3), did not affect the production
by the intact tissue with ulcers or that by normal tissue even at 50 µM. However, NS-398 dose dependently and significantly reduced the
increased PGE2 production by the
ulcerated tissue. It was evident that NS-398 is selective over somewhat
narrow concentrations. However, the effect of indomethacin on ulcerated
tissue was more potent than that of NS-398, with the inhibition being
~70% by indomethacin and ~50% by NS-398 at 25 µM, and ~80%
by indomethacin and ~65% by NS-398 at 50 µM.
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COX-2 expression in ulcer base. We examined the expression of COX proteins in the rat gastric tissue by means of Western blotting (Fig. 3A). Anti-COX-1 and anti-COX-2 antibodies reacted with the respective standard proteins without any cross-reaction. In the normal gastric tissue, one protein (70 kDa) was recognized by anti-COX-1 antibody. No proteins were detected by anti-COX-2 antibody. The same results were obtained for the intact tissue of the stomach with ulcers. However, in the ulcerated tissue, anti-COX-2 antibody reacted with one protein, ~70 kDa in weight, whose relative mobility was apparently identical to that of the standard COX-2 protein. One immunoreactive protein corresponding to COX-1 was also detected.
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Regulation of COX-2 mRNA expression.
It is well known that IL-1 and TNF- induce COX-2 expression and
that TGF-1 modulates COX-2 expression (6). Therefore, we first
examined whether or not the expression of IL-1, TNF-, and
TGF-1 mRNAs was induced in the ulcer base (Fig. 5).
Their mRNAs were not detected in either the mucosa of the normal
stomach or the intact mucosa of the stomach with ulcers. However, all three mRNAs were expressed in the ulcerated tissue on
day
0. The levels of mRNA expression
remained nearly constant until day
7 and decreased thereafter. The rate
of the decrease in TGF-1 mRNA expression after
day 7 was less than that of the decreases in IL-1 and TNF-.
|
, TNF-, and TGF-1 are involved
in COX-2 mRNA expression in the ulcer base, we antagonized the actions
of IL-1, TNF-, and TGF-1 in a culture of the isolated ulcer
base. After the isolated base was incubated for 48 h in the presence of
the indicated additives, the expression of COX mRNAs and
PGE2 production was determined. We
confirmed that the IL-1 receptor antagonist (2 µg/ml), anti-TNF-
antibody (5 µg/ml), anti-TGF-1 antibody (5 µg/ml), and
anti-TGF- receptor II antibody (4 µg/ml) completely inhibit the
cell responses to 2 ng/ml IL-1, 5 ng/ml TNF-, 10 ng/ml TGF-1,
and 10 ng/ml TGF-1, respectively (data not shown).
In both the ulcer bases isolated on
day 3 (in the early phase of ulcer healing) and
day
10 (in the late phase), treatment with
IL-1 receptor antagonist dose dependently caused decreases in COX-2
mRNA expression and PGE2
production (Fig. 6). The inhibitory effect of IL-1
receptor antagonist was more potent on
day 3 than on day
10. IL-1 receptor antagonist at 4 µg/ml significantly reduced COX-2 mRNA expression and
PGE2 production by 43.1 ± 6.6 and 33.2 ± 6.7% on day
0 and by 25.0 ± 7.9 and 22.7 ± 8.4% on day
10, respectively, compared with the
corresponding control. However, IL-1 receptor antagonist failed to
inhibit COX-1 mRNA expression on days
3 and 10.
|
antibody, similar results were obtained
(Fig. 7). In the ulcer base isolated on
day
3, COX-2 mRNA expression and
PGE2 production were inhibited by
anti-TNF- antibody in a dose-dependent manner, with the inhibition
by the antibody at 10 µg/ml being 34.7 ± 7.4 and 30.6 ± 6.4%, respectively. In the base isolated on
day
10, anti-TNF- antibody dose
dependently reduced COX-2 mRNA expression and
PGE2 production, but the
significant effect was observed only in the inhibition (17.9 ± 6.1%) of COX-2 mRNA expression at 10 µg/ml anti-TNF- antibody.
COX-1 mRNA expression was not affected by anti-TNF- antibody.
|
1 antibody promoted COX-2 mRNA
expression (Fig. 8). The antibody at 10 µg/ml
significantly increased COX-2 mRNA expression by 27.4 ± 4.4 and 46.1 ± 13.1% above the corresponding
control on days
3 and
10, respectively. Along with the
increase in COX-2 mRNA expression,
PGE2 production was also
stimulated. Anti-TGF-1 antibody at 10 µg/ml increased the
production by 23.7 ± 7.3 and 34.1 ± 9.6% above the control on days
3 and
10, respectively. In addition,
anti-TGF- receptor II antibody at 4 µg/ml also significantly
enhanced COX-2 mRNA expression and
PGE2 production by 132.1 ± 7.9 and 130.6 ± 9.0%, respectively, in the base isolated on
day
10. However, the expression of COX-1
mRNA was not affected by anti-TGF-1 antibody or anti-TGF- receptor II antibody.
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production but has no
effect on the production of other inflammatory proteins, such as IL-6
(30, 31). In addition, 10 µM FR-167653 did not affect COX-2 mRNA
expression in phorbol ester-stimulated fibroblasts (data not shown).
FR-167653 significantly suppressed the expression of both IL-1 and
TNF- mRNAs in a dose-dependent manner but had no effect on the
expression of TGF-1 or COX-1 mRNAs. COX-2 mRNA expression was
significantly reduced by the compound, in association with the
decreases in the expression of IL-1 and TNF- mRNAs. The
inhibition by 10 µM FR-167653 of the expression of IL-1, TNF-,
and COX-2 mRNAs was 62.0 ± 8.8, 35.7 ± 5.7, and 55.9 ± 3.0%, respectively. Furthermore, FR-167653 also dose dependently
inhibited PGE2 production, with
the inhibition at 10 µM being 42.4 ± 3.9%.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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These results clearly indicate that the expression of COX-2 mRNA and protein is induced only in the ulcerated gastric tissue in rats and that the level of COX-2 mRNA decreases with ulcer healing. These are consistent with the recent findings by Mizuno et al. (16) concerning mice. As reported previously by Szelenyi et al. (23) and us (29), PGE2 production was significantly elevated in the ulcerated tissue, compared with that in the intact tissue, and the increased production returned to the normal level with ulcer healing. The change in PGE2 production was well associated with COX-2 mRNA expression. In fact, the increased PGE2 production in the ulcerated tissue, but not the production in other tissue, was dose dependently inhibited by NS-398, a selective inhibitor of COX-2 (3). In addition, we (21) confirmed that indomethacin inhibits PGE2 production in both intact and ulcerated tissue, while NS-398 reduces production only in ulcerated tissue, after the drugs are administered to the rats with gastric ulcers. The inhibition of COX-2 mRNA expression in the isolated ulcer base also leads to the decrease in PGE2 production. Accordingly, these results indicate that COX-2 might contribute to the elevation of PGE2 production in the ulcerated tissue. In addition, COX-1 might also be involved in PGE2 production in the ulcerated tissue, because COX-1 protein and mRNA were present in the ulcerated tissue and indomethacin more potently reduced PGE2 production in the ulcerated tissue than NS-398 did.
Moreover, we defined the cellular localization of COX-2 protein. There were no immunoreactive signals for COX-2 protein in the intact mucosa around the ulcer base or the mucosa of normal rats, but strong immunoreactivity for COX-2 protein was found in fibroblasts, monocytes/macrophages, and granulocytes in the upper portion of the ulcer base. These findings are strongly supported by the results of Western and Northern blot analyses. In vitro studies (6, 15) have revealed the induction of COX-2 in these cell types in response to various stimuli. However, it remains unknown why COX-2 protein is enriched only in the cells present in the upper portion of the base, although fibroblasts, monocytes/macrophages, and granulocytes also exist in other portions of it. It is known that conventional NSAIDs inhibit PGE2 production in the ulcerated tissue, thereby impairing ulcer healing (11, 13, 23, 29). Recently, Mizuno et al. (16) reported that NS-398 prevents ulcer healing in mice. Although PG production was not examined after the administration of NS-398, Mizuno et al. (16) claim that selective inhibition of COX-2 activity may cause a delay in the healing. Taken together with the present results, it is suggested that these COX-2-expressing cells in the ulcer base may play a key role in the healing of gastric ulcers. The increased PGs will most likely exert various effects only in and around the ulcer base, because PGs act within a small area due to their short half-lives. One such effect is an increase in blood flow around gastric ulcers (7).
COX-2 mRNA expression was strongly induced by gastric ulceration, and the increased expression decreased with ulcer healing (i.e., degeneration of the ulcer base). The COX-2-expressing cells are fibroblasts, monocytes/macrophages, and granulocytes, which are infiltrated into the ulcerated tissue to form granulation. The numbers and proportions of such cells in the ulcer base decreased with ulcer healing. Accordingly, it is considered that growth factors and cytokines stimulating the migration and proliferation of these cells, such as fibroblast growth factor and IL-8, may be involved in COX-2 expression.
We report here that COX-2 mRNA expression is locally regulated in the
ulcer base. Our results indicate that IL-1 and TNF- might stimulate
COX-2 mRNA expression in the base. IL-1 and TNF- are typical
COX-2 inducers in a variety of cell types, including fibroblasts,
monocytes/macrophages, and granulocytes (6, 15). In fact, we also
confirmed that IL-1 and TNF- mRNAs are expressed only in the
ulcerated tissue. Similarly, Kinoshita et al. (9) reported that IL-1
is induced by gastric ulceration. In addition, the IL-1 type I receptor
might be implicated in the action of IL-1 on COX-2 mRNA expression,
because IL-1 receptor antagonist is known to bind more preferably to
the type I receptor than the type II receptor (22). In contrast,
TGF-1 might negatively regulate COX-2 mRNA expression in the ulcer
base, through the partial mediation of the TGF- type II
receptor. In this study, TGF-1 mRNA expression was
found only in ulcerated tissue, as reported by Tominaga et al. (25).
However, TGF- is reported to exert differential effects on COX-2
mRNA expression in vitro. In endotoxin-activated macrophages, TGF-
attenuates COX-2 mRNA expression (18), while it enhances expression in
mitogen-stimulated fibroblasts (5). At present, the reason why TGF-
reduces COX-2 mRNA expression in the base remains unclear. Because it
is well known that TGF- serves as a strong immunosuppressor (14),
the inhibitory effect of TGF- toward inflammatory cells may be more potent than its stimulatory effect toward fibroblasts in the ulcer base. Our results also suggest that, in the local regulation of COX-2
mRNA expression, the contribution of IL-1 and TNF- to the induction
is greater in the early phase of ulcer healing than in the late phase,
while the inhibitory action of TGF-1 is more potent in the late
phase than in the early phase. The levels of IL-1 and TNF- mRNA
expression on day
10 were reduced to ~50% of those on
day
3. Because it is suspected that
factors other than IL-1, TNF-, and TGF-1 might also be
involved in COX-2 mRNA expression in ulcerated gastric tissue,
functional interaction between them should be considered.
Among the factors stimulating COX-2 mRNA expression, the proportion of
IL-1 and TNF- may be high in the early phase but low in the late
phase. In contrast, the expression of TGF-1 mRNA was not largely
different between days
3 and
10. The ratio of stimulatory factors,
including IL-1 and TNF-, to TGF-1 may be crucial in the
expression of COX-2 mRNA. In general, the effects of inhibitors and
antagonists are known to be attenuated with increases in stimulating
activities. Namely, when the ratio is large in the early phase, the
effects of the stimulating factors may be strong, so that expression of the inhibitory effect of TGF-1 may be slight. In contrast, TGF-1 may reduce COX-2 mRNA expression more strongly, as the stimulating factors decrease with ulcer healing.
In conclusion, these results indicate that COX-2 protein is highly
localized in fibroblasts, monocytes/macrophages, and granulocytes in
the base of gastric ulcers in rats and that COX-2 mRNA expression might
be regulated positively by IL-1 and TNF- and negatively by
TGF-1.
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ACKNOWLEDGEMENTS |
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We thank N. J. Halewood for critical reading of the manuscript and M. Shimose, M. Yoshida, M. Ishikawa, N. Kobayashi, and H. Yamada for technical assistance.
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FOOTNOTES |
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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.
Address for reprint requests: S. Takahashi, Dept. of Applied Pharmacology, Kyoto Pharmaceutical Univ., Misasagi, Yamashina, Kyoto 607-8414, Japan.
Received 25 March 1998; accepted in final form 27 July 1998.
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Abstract
Introduction
Methods
Results
Discussion
References
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