Introduction
DKT, one of Japan’s most frequently prescribed Kampo formulas, decreases
intestinal dysmotility and inflammation and cures cold sensations in the abdomen,
mainly in the umbilical sections [1 ]
[2 ]. DKT comprises the following crude
ingredients: processed ginger, ginseng, zanthoxylum fruit, and malt sugar. DKT
increases blood flow in the superior mesenteric arteries in human and rat intestinal
mucosa [3 ]
[4 ], and it improves small intestinal motility in guinea pigs by
activating the TRPV1 [5 ]
[6 ]. A previous study has shown that DKT
increases the levels of gastrointestinal hormones and neuropeptides such as motilin,
vasoactive intestinal peptide, and CGRP [7 ].
Additionally, DKT increases mucosal blood flow in the rat colon by upregulating
adrenomedullin and activating the TRPA1 in the epithelium [4 ]
[8 ].
These findings suggest that an increase in mucosal blood flow in the
gastrointestinal tract is responsible for the clinical effects of DKT against cold
sensations and dysmotility in the abdomen. However, the underlying mechanism for the
activation of TRPA1 and TRPV1 in sensory nerves that leads to an increase in
DKT-induced GMBF has not been elucidated yet. This study aimed to determine whether
DKT facilitates an increase in GMBF in the rat stomach through activation of TRPV1-
and TRPA1-expressing sensory nerves.
Results
First, we studied the vasoactive effects of TRPV1 and TRPA1 activators on rat gastric
mucosa. The TRPV1 activator capsaicin (10 mmol/L) induced an
increase in GMBF, which peaked after 5 min of mucosal application of
capsaicin and remained at sub-maximum levels until 15 min; following this,
the responses returned to basal levels ([Fig.
1a ]). The TRPA1 activator allyl isothiocyanate (AITC, 10 mmol/L)
induced a rapid increase in GMBF (251.1±20.2%), which gradually
decreased to baseline values after 30 min of application ([Fig. 2a ]). The TRPV1 blocker BCTC
(2.7 mmol/L) clearly inhibited GMBF in response to capsaicin;
however, the TRPA1 blocker A-967079 (10 mmol/L) did not show similar
effects ([Fig. 1 ]). In contrast, the TRPA1
blocker A-967079 (10 mmol/L), but not BCTC, completely suppressed
GMBF in response to AITC ([Fig. 2 ]). These
blockers had no activity of their own.
Fig. 1 Effects of TRPV1 blocker BCTC and TRPA1 blocker A -967079 on
GMBF induced by the mucosal application of capsaicin in rat stomachs.
(a ) Time course of GMBF response to capsaicin (10 mmol/L)
in rat stomachs is shown. BCTC (2.7 mmol/L) was applied to the
chamber for 50 min starting at 20 min prior to capsaicin
(10 mmol/L) application. Capsaicin was topically applied to the
mucosa for 30 min. The data are expressed as a% increase in
basal values and represented as mean±S.E. of values every 5 or
10 min for 4 rats. *p<0.05 was considered
statistically significant when compared to the control group using
Student’s t-test. (b ) Maximal GMBF response induced by the
mucosal application of capsaicin (10 mmol/L) in the presence of BCTC
(2.7 mmol/L) or A-967079 (10 mmol/L). The data are expressed
as a% increase in basal values and represented as mean±S.E.
for 4 rats. *p<0.05 was considered statistically significant
when compared to the control group using Student’s t-test. Note that
an increased GMBF in response to capsaicin (10 mmol/L) was almost
completely inhibited by BCTC (2.7 mmol/L) but not A-967079
(10 mmol/L).
Fig. 2 Effects of TRPA1 blocker A-967079 and TRPV1 blocker BCTC on
gastric mucosal blood flow (GMBF) induced by the mucosal application of
allyl isothiocyanate (AITC) in rat stomachs. (a ) Time course of GMBF
response to AITC (10 mmol/L) in rat stomachs is shown. A-967079
(10 mmol/L) was applied to the chamber for 50 min starting
at 20 min prior to AITC (10 mmol/L) application. AITC was
topically applied to the mucosa for 30 min. The data are expressed
as a% increase in basal values, and they represent mean±S.E.
of values every 5 or 10 min for 4–7 rats.
*p<0.05 was considered statistically significant using
Student’s t-test. (b ) Maximal GMBF response induced by
mucosal application of AITC (10 mmol/L) in the presence of BCTC
(2.7 mmol/L) or A-967079 (10 mmol/L). The data are expressed
as a% increase in basal values and represented as mean±S.E.
for 4–7 rats. ***p<0.001 was
considered statistically significant compared to the control group using
Student’s t-test. Note that an increased GMBF in response to AITC
(10 mmol/L) was completely inhibited by A-967079 (10 mmol/L)
but not BCTC (2.7 mmol/L).
Intragastric administration of DKT (360, 720, and 1440 mg/mL) induced gastric
hyperemic responses in a concentration-dependent manner, and a significant effect
was observed at concentrations of 720 mg/mL or higher ([Fig. 3a ]). The maximal responses of GMBF to
10 min of intragastric application of DKT at 360, 720, and
1440 mg/mL were 128.9±6.2%,
136.0±10.4%, and 162.9±8.5%, respectively ([Fig. 3b ]). Mucosal application of the vehicle
(distilled water) did not increase GMBF ([Fig. 3a,
b ]). GMBF reached its maximum value after administration of
1440 mg/mL of DKT; thus, this concentration was used in subsequent
experiments to examine the effects of various agents on GMBF in response to DKT.
Interestingly, the maximum GMBF levels after administration of
1440 mg/mL of DKT were similar to that observed after administration
of capsaicin (1 mmol/L) into the rat stomach (Supporting Information
Fig. 1b).
Fig. 3 Effect of the mucosal application of daikenchuto (DKT: 360,
720, and 1440 mg/mL) on GMBF in rat stomachs. (a ) Time course
of GMBF response to DKT. The data are expressed as a% increase in
baseline values, and they represent the mean±SE obtained every 2 or
10 min for 4–6 rats. *p<0.05 was considered
statistically significant compared to the control group using ANOVA with
Dunnett’s test. (b ) Maximum response of GMBF induced by the
mucosal application of DKT (360, 720, and 1440 mg/mL). The data are
expressed as a% increase in baseline value and represented as
mean±SE for 4–6 rats. *P<0.05 and
* *p<0.01 were considered statistically
significant compared to the control group using ANOVA with Dunnet’s
test. Note that GMBF response to DKT increases in a concentration-dependent
manner.
Intragastric pre-application of the TRPV1 blocker BCTC (2.7 mmol/L) alone did
not affect GMBF. The mucosal application time of DKT (1440 mg/mL) was
extended from 10 to 30 min to investigate further the effects of initial and
late periods during DKT application in rats ([Fig.
4a ]). A significant increase in GMBF was observed in response to DKT
(1440 mg/mL) for 30 min, and the gastric hyperemic responses
induced by DKT (1440 mg/mL) after 10 and 30 min of
application were 216.5±15.4% and 178.0±20.1%,
respectively ([Fig. 4b, c ]). The increase in
GMBF in response to DKT (1440 mg/mL) was decreased by BCTC
(2.7 mmol/L) during the late period but not during the initial
period. The values of GMBF after 10 and 30 min of mucosal application of DKT
in the presence of BCTC (2.7 mmol/L) were
203.3±34.3% and 150.6±5.3%, respectively. In
contrast, the increase in GMBF in response to DKT (1440 mg/mL) in
the initial and late periods was decreased by the TRPA1 blocker A-967079
(10 mmol/L) ([Fig. 4a ]). The
GMBF values after 10 and 30 min of mucosal application of DKT in the
presence of A-967079 (10 mmol/L) were 138.3±11.1%
and 139.1±14.3%, respectively ([Fig. 4b, c ]). Intragastric pre-application of A-967079
(10 mmol/L) alone did not affect GMBF. These results suggest that
intragastric DKT induces a significant increase in GMBF via activation of
TRPV1 and TRPA1, which may be due to the gastric epithelial cell expressing TRPA1
in
the initial phase followed by the TRPA1- and TRPV1 co-expressing nerve fiber in the
late phase.
Fig. 4 Effect of TRPV1 blocker BCTC and TRPA1 blocker A-967079 on GMBF
induced by the mucosal application of DKT in rat stomachs. (a ) Time
course for analysis of GMBF response to DKT (1440 mg/mL) in rat
stomachs treated with either A-967079 (10 mmol/L) or BCTC
(2.7 mmol/L). The data are expressed as a% increase in
baseline values, and they represent the mean±SE values obtained
every 5 or 10 min for 4–5 rats. *P<0.05 was
compared with the vehicle using Student’s t-test. (b ) GMBF
after 10 min of DKT administration (1440 mg/mL) in rat
stomachs treated with either A-967079 (10 mmol/L) or BCTC
(2.7 mmol/L). (c ) GMBF after 30 min of DKT
(1440 mg/mL) administration in rat stomachs treated with either
A-967079 (10 mmol/L) or BCTC (2.7 mmol/L). The data are
expressed as a% increase in baseline values and represented as
mean±SE for 4–5 rats.
* *p<0.01 was compared with the vehicle
using Student’s t-test. Note that the increased GMBF in response to
DKT is obviously suppressed by A-967079 but is not partially suppressed by
BCTC in the initial periods.
As expected, it was observed that the increased GMBF in response to DKT was inhibited
by the TRPA1 blocker A967079 in both initial and late phases, while the TRPV1
blocker BCTC inhibited the increased GMBF in response to DKT only in the late phase
of the experiment.
A significant increase in GMBF in response to DKT (1440 mg/mL) for
30 min was observed in animals treated with the vehicle (distilled water),
and the gastric hyperemic responses induced by DKT (1440 mg/mL) after 10 and
30 min of application were 144.5±31.6% and
143.0±10.4%, respectively ([Fig.
5a ]). The increase in GMBF values in response to mucosal application of
DKT (1440 mg/mL) in the deafferentation of capsaicin-sensitive
sensory fibers were 122.5±11.1% and 103.4±6.8%,
after 10 and 30 min, respectively, implying a significant decrease in the
late phase, but not in the initial phase ([Fig. 5b,
c ]). GMBF response to DKT after removing mucosal application was similar
to that observed in BCTC (2.7 mmol/L)-treated animals as shown in
[Fig. 4a ]. Thus, these results suggest
that intragastric DKT produces a significant increase in GMBF via activation
of TRPV1 and TRPA1, which are expressed in capsaicin-sensitive sensory nerves,
especially in the late phase, but not in the gastric epithelial cells of
anesthetized rats. Numerous TRPV1-immunoreactive axons were found around the
arterioles in the submucosal layer of the stomach ([Fig. 6a ]). TRPA1-immunoreactive axons were also observed around the
arterioles in the submucosal and circular muscle of the stomach ([Fig. 6b ]). Additionally, TRPA1-immunoreactive
cell bodies were also present in the myenteric plexus of the stomach. Interestingly,
capsaicin-pretreatment to produce deafferentation of capsaicin-sensitive sensory
fibers in rats abolished TRPA1-immunoreactivities in the submucosa but not in the
circular muscle of the stomach ([Fig. 6d ]).
However, TRPV1-immunoreactivities were abolished entirely in the submucosal and
mucosal layers of the stomach ([Fig. 6c ]).
Moreover, TRPA1-immunoreactive cells were also confirmed in the gastric mucous of
both the control and capsaicin-pretreatment groups. This was also present in the
mucosal cell of the stomach after capsaicin-pretreatment ([Fig. 6b and d ]).
Fig. 5 Effect of deafferentation of capsaicin-sensitive sensory fiber
on GMBF induced by the mucosal application of DKT in rat stomachs. DKT
(1440 mg/mL) was applied topically to the mucosa for 30 min,
and the stomach was perfused with saline before and after application.
(a ) Time course for analysis of GMBF in response to DKT
(1440 mg/mL) in capsaicin-pretreated rats. Data are expressed as
the% increase in baseline values and represented as mean±SE
values obtained every 5 or 10 min for 4–8 rats.
*p<0.05 was considered statistically significant compared to
the control group using Student’s t-test. b ) and c )
GMBF after 10 and 30 min of mucosal application of DKT
(1440 mg/mL) in capsaicin-pretreated rats, respectively. Data are
expressed as the% increase in baseline values and represented as
mean±SE for 4–8 rats.
* *p<0.01 was considered statistically
significant when they compared to the control group using Student’s
t-test.
Fig 6 Alteration in the distribution of immunoreactivities of TRPV1
and TRPA1 in the cross-section of rat stomach in between control and
capsaicin-pretreatment. The immunoreactive fibers of TRPV1 (a ) and
TRPA1 (b ) were found in the submucosa, myenteric plexus, and
muscularis. TRPV1- and TRPA1-expressing fibers were observed around the
submucosal arteriole and venule bundles (arrows) (a , b ). Note
that TRPA1-immunoreactive cell bodies are present in the myenteric plexus
(arrowheads), and the TRPA1-immunoreactivity was also confirmed in the
mucous cells of the gastric glands (2-headed arrow). The scale bar is
60 µm (a , b ). No TRPV1-expressing fiber is
found in the capsaicin-pretreated rat stomach (c ). Notice that
TRPA1-expressing fibers around the submucosal arteriole and venule bundles
are not observed in capsaicin-pretreated rats (d ), although the
TRPA1-immunoreactivity is observed in the myenteric plexus (arrows) and
mucous cells in the bottom of gastric glands after capsaicin pretreatment
(2-headed arrow). MU, mucous membrane; CM, circular muscle. The scale bar
corresponds to 120 µm (c , d ). Two transverse
sections of tissues obtained from 5 normal and capsaicin-pretreated rats are
stained by immunohistochemical methods. The typical photos are shown in this
figure.
Discussion
In the present study, both the initial and late phases of increased GMBF in response
to DKT were inhibited by the TRPA1 blocker A967079. However, only the late phase was
inhibited by the TRPV1 blocker BCTC. In the capsaicin-pretreated rats, the late
phase of the DKT-increased GMBF was abolished. Additionally, the immunohistochemical
study clarified that TRPV1- and TRPA1-coexpressing nerve fibers were abolished in
the submucosal layer in the capsaicin-pretreated rats. Taken together, these TRPV1-
and TRPA1-coexpressing sensory nerve fibers are involved in the late phase of
DKT-induced GMBF. Therefore, it was observed that an increase in GMBF in response
to
DKT was significantly but not completely inhibited by the deafferentation of
capsaicin-sensitive sensory fiber after capsaicin-pretreatment in the late phase
(15–30 min) than in the initial phase
(0–15 min).
In contrast, some myenteric TRPA1-expressing neurons in the smooth muscle layer
remain in the capsaicin-pretreated rats, and they are thought to be the intrinsic
primary afferent neurons. Therefore, it is supposed that those neurons that express
TRPA1 but not TRPV1 are involved in the initial phase of DKT-induced GMBF.
Interestingly, TRPA1-expressing mucosal cells were also observed in gastric glands.
It is reported that the vasodilator Adrenomedullin is released from TRPA1-expressing
intestinal epithelium in response to AITC and DKT in the rat intestine [8 ]. Therefore, the initial phase of DKT-induced
GMBF may be attributed to vasodilators such as Adrenomedullin from the
TRPA1-expressing mucosal cells. Taken together, the initial phase of increased GMBF
in response to DKT may be due to the activation of TRPA1 that is expressed in
sensory neurons and gastric epithelial cells.
It is well known that gastric hyperemic responses increase after the first
application of capsaicin. However, the response to the second application of
capsaicin is approximately one-third of that of the first application, suggesting
that mucosal hyperemic responses are desensitized by repeated application of
capsaicin [9 ]
[10 ]. Additionally, the repeated application of AITC induces
desensitization of GMBF, suggesting that TRPA1 can be desensitized by AITC [11 ]. In contrast, the response to the second
application of DKT was almost as strong as that to the first application (Supporting
Information Fig. 1b), suggesting that the increased GMBF in response to DKT may be
attributed to not only the activation of TRPV1 and TRPA1 but also the blockage of
2-pore-domain potassium channels (KCNKs). For example, Zanthoxylum fruit was one of
the ingredients of DKT, a known activator of TRPA1 and TRPV1 (actions resembling
those of processed ginger compounds) and a blocker of the potassium leak channels,
such as KCNKs [12 ]. Additionally, KCNKs
maintain the cell’s resting potential and regulate cellular excitability in
exciting cells such as neurons. Therefore, DKT may be blocking KCNKs in
capsaicin-sensitive sensory fibers by the action of Zanthoxylum fruit, which leads
to increased membrane excitability. There is a decreased threshold for additional
exogenous stimuli such as DKT [13 ]. Although
it has been shown that capsaicin and AITC activated TRPV1 and TRPA1, respectively,
they also inhibited nerve conduction via voltage-gated
Na+ channels in a manner independent of TRPV1 and TRPA1 activation
in the sciatic nerve of frogs [14 ]
[15 ]. The inhibition of nerve conduction
required about 20–100 times higher concentration of capsaicin
(200 µM) and AITC (2 mM) than the activation of TRPV1
(1 µM) and TRPA1 (100 µM) in the DRG neurons [16 ]
[17 ].
This study did not observe whether nerve conduction was inhibited by capsaicin and
AITC, which might have increased GMBF. The increased GMBF in response to capsaicin
and AITC was induced by TRPV1- and TRPA1-co-expressing sensory nerves.
It is shown that the TRPV1 blocker BCTC also inhibited the activation of TRPM8 in
response to menthol in TRPM8-transfected HEK293 cells in vitro, so we studied
whether or not the increased GMBF induced by mucosal application to the TRPM8
activator menthol (100 mM) or WS-12 (0.2–0.8 mM) was
observed in rat stomachs [18 ]. However, it was
not observed that menthol and WS-12 induced the increased GMBF, suggesting that
increased GMBF was not attributed to the activation TRPM8.
Previously, it has been reported that 1) numerous TRPV1-immunoreactive axons are
found around the arteries in the lower mucosa of the rat stomach [19 ] and 2) capsaicin stimulates the TRPV1 nerve
fibers to release CGRP [20 ] and nitric oxide
(NO) [10 ]
[21 ]. Additionally, TRPV1 and TRPA1 are co-expressed in the extrinsic
sensory nerves [22 ]. These data led us to
hypothesize that activating TRPA1- and TRPV1-expressing sensory nerves by DKT
increased GMBF. Indeed, in this study, an increased GMBF induced by DKT in the
deafferentation of capsaicin-sensitive sensory fibers in rats was mainly and
strongly inhibited in the late phase. Further studies are required to elucidate the
unknown vasorelaxant mediators to clarify the mechanism underlying the increased
GMBF in response to DKT. Kampo medicines are used to treat various diseases in
Japan. DKT is one of the most frequently used Kampo medicines, which is clinically
effective in treating cold sensations and dysmotility in the abdomen. Although
previous studies have shown the effects of DKT in improving these symptoms [1 ]
[5 ],
our results indicate that DKT stimulates TRPV1 and TRPA1 on sensory nerves to
promote gastric mucosal circulation.
In conclusion, the present study showed that the mucosal application of DKT increased
GMBF via TRPA1- and TRPV1-expressing sensory nerves. For the first time, we
demonstrated that the initial and the late phases of the increased GMBF in response
to DKT were inhibited by the TRPA1 blocker A967079. However, only the late phase was
inhibited by the TRPV1 blocker BCTC. In the capsaicin-pretreated rats, the late
phase of the DKT-increased GMBF was abolished. Additionally, the immunohistochemical
study clarified that TRPV1- and TRPA1-coexpressing nerve fibers were abolished in
submucosal layers in the capsaicin-pretreated rats. Taken together, these TRPV1- and
TRPA1-coexpressing sensory nerve fibers are involved in the late phase of
DKT-increased GMBF.
Materials and Methods
Animals
Male Sprague-Dawley rats weighing 180–220 g were purchased from
Japan SLC Co., Ltd. Animals were housed under controlled environmental
conditions (temperature: 24±2°C and lights on from 7:00 am to
7:00 pm), and they were fed commercial rat chow MF (Oriental Yeast,
Tokyo, Japan). The animals were kept in individual cages with raised mesh
bottoms to prevent coprophagia and were deprived of food but allowed free access
to tap water for 18 h before the experiments. Animal experiments were
performed in compliance with the “Guiding Principles for the Care and
Use of Laboratory Animals” approved by the Japanese Pharmacological
Society. The study procedure was approved by the Ethical Committee on Animal
Care and Animal Experimentation of Josai International University (#1800051,
registered on March 13, 2018, and # 1900011, registered on March 1, 2019). A
minimum number of animals were used for meaningful interpretation of data.
Materials
The following drugs were used: AITC (purity>95%, Tokyo Chemical
Industry, Saitama, Japan), atenolol (purity>98%, Wako, Osaka,
Japan), capsaicin (purity>60%, Wako, Osaka, Japan),
DL-isoproterenol (purity>98%, Wako, Osaka, Japan), urethane
(ethyl carbamate, purity>98%, Wako, Osaka, Japan), A-967079
(purity>98%, Tocris Cookson, MN, USA), and BCTC
(purity>98%, Biomol, PA, USA). AITC was suspended in
0.1% Tween 80 (purity>75%, Wako, Osaka, Japan)-saline
for mucosal application. Capsaicin was dissolved in Tween 80-ethanol solution
(10% ethanol (Wako, Osaka Japan), 10% Tween 80, and 80%
saline (Otsuka, Tokushima, Japan) [23 ] for
subcutaneous (s.c.) injection, or it was suspended in 0.5% CMC (Wako,
Osaka, Japan) for mucosal application. DKT extract (Tsumura, Tokyo, Japan
& Co., Lot No. 2100100010) and maltose syrup powder (Tsumura, Tokyo,
Japan & Co., Lot No. 3020168) were used in this study. The composition
of the herbal DKT medicine is the following: (1) Japanese pepper (Zanthoxylum
fruit; peel of Zanthoxylum piperitum De Candolle), (2) processed ginger
(Zingiberis Siccatum Rhizoma; rhizome of Zingiber officinale Roscoe), (3)
ginseng (Ginseng radix; root of Panax ginseng C. A. Meyer), and (4)
maltose syrup powder, which was used as glue. The traditional medicine was made
by mixing the herbs in defined ratios by the Pharmacopoeia of Japan, followed by
extraction using hot water. Finally, the extract was made into a powder form
[12 ].
DKT extract powder was manufactured as an aqueous extract containing processed
ginger, ginseng, and zanthoxylum fruit in a ratio of 5:3:2. HPLC analysis showed
the presence of 6-gingesulfonic acid, hydroxy α -sanshool, hydroxy
β -sanshool, 6-gingerol, α-sanshool,
β -sanshool, γ -sanshool, and 6-shogaol. These are
the main compounds of zanthoxylum fruit and dry-processed ginger. (Supporting
Information Fig. S2). DKT was prepared by mixing DKT extract powder and maltose
syrup powder in a ratio of 1:8. The DKT extract was suspended in distilled water
for intragastric (i.g.) application. Although the doses of DKT in the present
study (1440 mg/mL) were higher than the clinical doses used in
humans, previous studies in animals have shown that the relevant pharmacological
effects occur only at the experimental doses. A-967079 was dissolved in DMSO
(Wako) in saline, and the final concentration of DMSO was less than
5.0%. Other drugs were dissolved in saline with no organic solvents or
detergents. Drugs were administered at a volume of
0.5 mL/100 g of body weight in the case of
intraperitoneal (i.p.) and s.c. administration. Control animals received only
the vehicle.
GMBF measurement
The animals were anesthetized with urethane (1.25 g/kg, i.p.).
The stomach was exposed through a midline incision, delivered to the abdominal
surface by gentle traction on the spleen, and the pylorus was ligated. A 2-part
lucite chamber was used to maintain ex vivo conditions of the gastric
mucosa [24 ]. The 2 holes were cannulated
to perfuse the mucosa with saline (NaCl, 154 mmol/L) at
37°C at a 1 mL/min flow rate. The lucite base was
lowered over the animal, and the stomach was drawn through the center hole with
forceps. Then, the stomach was opened along the greater curvature, and the edges
were expanded by gently stretching the glandular mucosa. A plastic rim was then
applied and pressed down onto the mucosa, such that only the glandular mucosa
area was exposed [25 ]. The chamber was set
at the level of the abdominal wall so that the external wall of the stomach
remained inside the abdominal cavity. The body temperature was maintained at
approximately 37°C using an incandescent lamp. GMBF was measured using
laser-Doppler flowmetry (Model ALF-21N, Advance, Tokyo, Japan) and a touching
probe (diameter, 1 mm) on the surface of the corpus mucosa. After
stabilization of GMBF, the perfusion was discontinued, the luminal solution was
removed, and mucosa was exposed to 2 mL of DKT for 10 or 30 min.
After applying DKT, the mucosa was rinsed with saline, another 2 mL of
saline was instilled, and the perfusion was resumed. Changes in GMBF were
continuously monitored and recorded for 2 h test periods using a Power
Lab system (Model ML845; AD Instruments, Nagoya, Japan). Either the BCTC
(2.7 mmol/L, i.g.) or A-967079 (10 mmol/L, i.g.)
was given for 50 min, starting at 20 min before DKT application
[26 ]
[27 ]. Deafferentation of capsaicin-sensitive sensory fiber, which
means capsaicin-pretreatment, was performed 2 weeks before the experiment by
successive s.c. injections of capsaicin (20, 30, and 50 mg/kg)
once daily for 3 days [23 ]. All capsaicin
injections were given under ether anesthesia, and the rats were pretreated with
the β -adrenergic receptor agonist isoproterenol
(0.01 mg/kg, intramuscular (i.m.)) and the selective
β
1 -adrenergic antagonist atenolol
(0.01 mg/kg, i.m.) to counteract the respiratory impairment
associated with capsaicin. To verify the effectiveness of the treatment, we
placed a drop of capsaicin (0.1 mg/mL) in 0.5% CMC into
one eye of each rat, and the protective wiping movements were counted [28 ].
Immunostaining
The corpora of rat stomachs were fixed by immersing in 4% fresh
paraformaldehyde in 0.1 mol/L phosphate butter (pH 7.4) at
4°C for 2 h. Subsequently, they were washed thrice with PBS and
cryoprotected overnight in 0.1 mol/L phosphate buffer containing
20% sucrose. The tissues were frozen in optimal cutting temperature
(OCT) compound mounting medium (Sakura Finetek, Japan) and sectioned into 60
μm thick sections using a cryostat (Leica, Hemel Hempstead, UK). The
tissue sections were thawed and mounted onto MAS courting-slides (MSA-15,
Matsunami Glass, Japan). Prior to staining, the slide-mounted sections were
incubated in PBS containing 0.3% hydrogen peroxide
(purity=30%, Wako, Osaka, Japan) for 30 min to quench
endogenous peroxidase activity and washed with PBS. The sections were
successively incubated in 10% normal donkey serum containing
0.2% Triton X-100, 0.1% sodium azide (rehydration according to
instruction to yield 100% serum, Jackson ImmuneResearch Laboratories,
PA, USA) in PBS for 1 h, and a polyclonal anti-TRPV1 antibody (1:30,000,
Neuromics, MN, USA) or polyclonal anti-TRPA1 antibody (1:120,000; Abcam,
Australia) for 40 h at 25℃. TRPV1 immunoreactivity was detected
using a polyclonal antiserum raised in a rabbit against the carboxy terminus of
mouse TRPV1. In contrast, TRPA1 immunoreactivity was detected using a polyclonal
antiserum raised in rabbits against the fourth cytoplasmic loop of rat and mouse
TRPA1. After washing with PBS, the sections were incubated with biotinylated
donkey anti-rabbit immunoglobulin G (1:400; Jackson ImmunoResearch Laboratories,
PA, USA) at room temperature for 90 min. After further washes with PBS 3
times, the sections were incubated in streptavidin-biotin-complex (1:5 in PBS,
Vectastain Elite ABC kit, Vector Laboratories, CA, USA) at room temperature for
1 h, followed by incubation with fluorescein tyramide (1:75; TSA kit;
Perkin Elmer Life Sciences, MA, USA). In control experiments, either the TRPV1
antibody or TRPA1 antibody was omitted from the staining procedure to verify
staining specificity. No immunolabeling was observed in any of the controls.
Each antiserum was diluted in PBS containing 0.2% Triton X-100 and
0.1% sodium azide.
Microscopy
Sections were viewed on either a Leica epifluorescence microscope (Leica
microsystems, Wetzlar, Germany) using Y3 (TRITC) and L4 (FITC) filter blocks or
an upright Olympus confocal laser scanning microscope (FV-1000, Tokyo, Japan)
system (FX-1000) with 20X and 40X Plan Neofluar objectives. A single tracking
mode was used for single FITC labeling in the latter. Sections were scanned in
the multi-tracking mode to avoid channel crosstalk when double labeling was
performed with FITC (488 nm) and TRITC (543 nm). Each image was
produced by projecting all stack slices onto a single plane. The final plates
were prepared using Adobe Photoshop.
Statistical analysis
The data are presented as the mean±SE for 4–8 rats per group.
Concentration-dependency was determined using analysis of variance. Statistical
analysis was performed using Dunnett’s multiple comparison test and
Student’s t-test on Sigma Plot 13.0 software (Systat) for Windows.
Statistical significance was set at p<0.05.
