Am J Physiol Gastrointest Liver Physiol 297: G348–G360, 2009.
First published June 4, 2009; doi:10.1152/ajpgi.90578.2008.
Localization of TRPV1 and contractile effect of capsaicin in mouse large
intestine: high abundance and sensitivity in rectum and distal colon
Kenjiro Matsumoto,1 Emi Kurosawa,2 Hiroyuki Terui,1 Takuji Hosoya,1 Kimihito Tashima,1
Toshihiko Murayama,2 John V. Priestley,3 and Syunji Horie1
1
Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, Togane, Chiba;
Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba,
Japan; and 3Neuroscience Centre, Institute of Cell and Molecular Science, Barts and The London School of Medicine and
Dentistry, Whitechapel, London, United Kingdom
2
Submitted 5 October 2008; accepted in final form 28 May 2009
vanilloid; immunohistochemistry; afferent nerve; substance P; neurokinin A
vanilloid type 1 (TRPV1)
channel, also referred to as the capsaicin receptor, is described
as a polymodal sensor of noxious heat (⬎43°C), low pH (pH ⬍
5.9), and inflammatory pain (12). In the gut, TRPV1 modulates
physiological functions such as motility, secretion, circulation,
and visceral nociception (8, 18, 19). Immunohistochemical
studies have demonstrated the presence of TRPV1 channels in
the gastrointestinal tract in the mucosa, submucosal layer, and
muscular layer in the guinea pig, mouse, and rat gastrointestinal tract (23, 31, 37). Therefore, TRPV1 has become an
attractive molecular target for pharmacological intervention in
gastroenterological research.
THE TRANSIENT RECEPTOR POTENTIAL
Address for reprint requests and other correspondence: K. Matsumoto, Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International
Univ., 1 Gumyo, Togane, Chiba 283-8555, Japan (e-mail: [email protected]).
G348
Capsaicin, the main pungent constituent in red peppers of
the genus Capsicum, stimulates TRPV1 channels, leading to
the activation of primary afferent neurons with unmyelinated
or thinly myelinated nerve fibers, the so-called capsaicinsensitive afferent neurons. Capsaicin has been utilized by
numerous investigators to activate afferent fiber endings in
studies of the visceral effects of TRPV1 in the gastrointestinal
tract, and it has been established that capsaicin-sensitive primary afferent neurons participate in the regulation of gastrointestinal motility (8). For example, capsaicin induces contractions in the guinea pig ileum (8) and relaxation in human small
and large intestines (7, 27, 28). Afferent nerves in the gut not
only send signals to the central nervous system but also provide
a local efferent-like effect by releasing neuropeptides. Capsaicin-sensitive nerve fibers that contain neuropeptides including
tachykinins and calcitonin gene-related peptide have been
identified in several mammalian species. In particular, tachykinins such as substance P and neurokinin A mediate the
excitatory effect of capsaicin on sensory neurons (8). Neurochemical and functional evidence indicates that tachykinins are
expressed in extrinsic and intrinsic primary afferent neurons
(15, 21).
The gastrointestinal tract contains two types of primary
afferent neurons: intrinsic primary afferent neurons with cell
bodies, processes, and synaptic connections in the gut wall, and
extrinsic primary afferent neurons with their cell bodies in
nodose and jugular ganglia or in dorsal root ganglia (16).
TRPV1 channels are found predominantly on the sensory
afferent nerve fibers implicated in pain transduction. TRPV1
nerve fibers in the gut appear to be predominantly extrinsic in
origin (22, 31, 37), but their existence in intrinsic neurons in
the gastrointestinal tract was also reported (3).
The TRPV1 channel has become an attractive target for
treatment of colonic and rectal disorders such as irritable bowel
syndrome and inflammatory bowel disease (2, 13, 39). However, most studies of the physiological roles of TRPV1 in
motor function in the intestine have been carried out mainly
using isolated guinea pig ileum. The effects of capsaicin and
the distribution of TRPV1 channels in the lower gastrointestinal tract remain largely unstudied. In particular, regional and
hence functional differences in TRPV1 among the rectum and
distal, transverse, and proximal colon have never been reported. In the present study, we investigated the immunohistochemical differences in the distribution of TRPV1 channels
and the contractile effect of capsaicin on smooth muscle in the
mouse rectum and distal, transverse, and proximal colon. We
also investigated the contractile mechanism of capsaicin in
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Society
http://www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Matsumoto K, Kurosawa E, Terui H, Hosoya T, Tashima K,
Murayama T, Priestley JV, Horie S. Localization of TRPV1 and
contractile effect of capsaicin in mouse large intestine: high abundance and sensitivity in rectum and distal colon. Am J Physiol
Gastrointest Liver Physiol 297: G348 –G360, 2009. First published
June 4, 2009; doi:10.1152/ajpgi.90578.2008.—We investigated immunohistochemical differences in the distribution of TRPV1 channels
and the contractile effects of capsaicin on smooth muscle in the mouse
rectum and distal, transverse, and proximal colon. In the immunohistochemical study, TRPV1 immunoreactivity was found in the mucosa,
submucosal, and muscle layers and myenteric plexus. Large numbers
of TRPV1-immunoreactive axons were observed in the rectum and
distal colon. In contrast, TRPV1-positive axons were sparsely distributed in the transverse and proximal colon. The density of TRPV1immunoreactive axons in the rectum and distal colon was much higher
than those in the transverse and proximal colon. Axons double labeled
with TRPV1 and protein gene product (PGP) 9.5 were detected in the
myenteric plexus, but PGP 9.5-immunoreactive cell bodies did not
colocalize with TRPV1. In motor function studies, capsaicin induced
a fast transient contraction, followed by a large long-lasting contraction in the rectum and distal colon, whereas in the transverse and
proximal colon only the transient contraction was observed. The
capsaicin-induced transient contraction from the proximal colon to the
rectum was moderately inhibited by an NK1 or NK2 receptor antagonist. The capsaicin-induced long-lasting contraction in the rectum
and distal colon was markedly inhibited by an NK2 antagonist, but not
by an NK1 antagonist. The present results suggest that TRPV1
channels located on the rectum and distal colon play a major role in
the motor function in the large intestine.
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
each segment of the lower gastrointestinal tract, with the
special reference to the involvement of the tachykinin NK1 and
NK2 receptors.
MATERIALS AND METHODS
Animals
Drugs
The drugs used in this study were acetylcholine chloride, tetrodotoxin (TTX), capsaicin, and atropine sulfate (Wako Pure Chemical
Industries, Tokyo, Japan), N-(4-tertiary butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC) (Biomol
International, Boechout, Belgium), NG-nitro-L-arginine methyl ester
(L-NAME), 1-(2-(5-fluoro-1H-indol-3-yl)ethyl)-4-methoxy-4-((phenylsulfinyl)methyl)piperidine (GR159897), substance P, neurokinin A
(Sigma Chemical, St. Louis, MO), N2-[(4R)-4-hydroxy-1-(1-methyl1H-indol-3-yl)carbony-1-L-prolyl]-N-methyl-N-phenylmethyl-3–2-(2naphtyl)-L-alaninamide (FK888), and 3-methyl-2-phenyl-N-[(1S)-1phenylpropyl]-4-quinolinecarboxamide (SB222200) (TocrisCookson, Bristol, UK).
Capsaicin was dissolved in ethanol prior to dilution in deionized
water. FK888, GR159897, SB222200, BCTC, and neurokinin A were
dissolved in DMSO prior to dilution in deionized water. Other drugs
were dissolved in deionized water with no organic solvent or detergent. The final concentrations of DMSO and ethanol in the bath were
less than 0.2 and 0.1%, respectively. The vehicles used had no
pharmacological effects on the tonus of preparations or the capsaicininduced contraction.
Isolated lower gastrointestinal tract and measurement of tension.
The preparation and measurement of contraction of segments of the
mouse rectum and distal, transverse, and proximal colon were performed as described previously (32). Each segment was removed and
placed in Krebs-Henseleit solution (in mM: 112.08 NaCl, 5.90 KCl,
1.97 CaCl2, 1.18 MgCl2, 1.22 NaH2PO4, 25.00 NaHCO3, and 11.49
glucose). A segment of each tissue, ⬃1.0 cm length, was set up under
a 0.7-g load in a 10-ml organ bath filled with Krebs-Henseleit
solution. The optimal resting tension was determined by preliminary
experiments. When the resting tension was 0.7 g, the maximum
reproducible contraction by capsaicin was obtained. The strips were
mounted in the longitudinal direction, and longitudinal muscle tension
was recorded. The bath was maintained at 37°C and continuously
bubbled with a mixture of 95% O2 and 5% CO2. Contraction was
recorded by use of an isotonic transducer (TD-112S, Nihon Kohden,
Tokyo, Japan), a balancing box (JD-112S, Nihon Kohden), and a
Powerlab system (AD Instruments, Castle Hill, Australia). At the start
of each experiment, the responsiveness to acetylcholine at 10 ␮M, a
submaximal concentration for acetylcholine-induced contraction, was
ascertained to evaluate the contractile effects of the capsaicin, substance P, and neurokinin A. After at least three stable contractions
were induced by 10 ␮M acetylcholine, the experiments were
performed.
AJP-Gastrointest Liver Physiol • VOL
In the first series of experiments investigating the noncumulative
concentration response to capsaicin, intestine segments were challenged only once with an individual dose of capsaicin (10 nM–10
␮M) to avoid the influence of sensitization or desensitization on the
contraction (10, 14). To investigate the possibility of desensitization
to capsaicin in the contractile responses, the intestine preparations
were incubated for 3 min with capsaicin (first treatment), then washed
three times with the Krebs-Henseleit solution for 5 min each. After
that, capsaicin at the same dose as the first treatment was added
(second treatment). Each response was expressed as a percentage of
the maximum contraction induced by 10 ␮M acetylcholine (% of
acetylcholine contraction).
Mechanism of capsaicin-induced contraction. In studies of the
effects of TTX, atropine, the nitric oxide synthase inhibitor L-NAME,
or TRPV1 antagonist BCTC on capsaicin-induced contraction, each
segment was preincubated with 300 nM of TTX for 10 min, 1 ␮M of
atropine for 10 min, or 100 ␮M of L-NAME for 10 min, or 1 ␮M of
BCTC for 45 min. In studies of the effects of the NK1 receptor
antagonist FK888 and NK2 receptor antagonist GR159897, each
segment was preincubated with 10 ␮M of FK888 or 3 ␮M of
GR159897 for 20 min. The drug treatment protocols were previously
described (4, 14). These drugs did not affect the baseline tension. Each
response was expressed as a percentage of the maximum contraction
induced by 10 ␮M acetylcholine (% of acetylcholine contraction).
Tissue preparation for immunohistochemistry. Segments of the
mouse rectum and distal, transverse, and proximal colon were removed, fixed by immersion in fresh 4% paraformaldehyde in 0.1 M
phosphate buffer for 2 h at 4°C, and washed three times with
phosphate-buffered saline (PBS). They were cryoprotected overnight
in 0.1 M phosphate buffer containing 20% sucrose. The tissues were
frozen in OCT (Sakura Finetek, Toyko, Japan) mounting medium, and
sectioned on a cryostat (Leica Instruments, Nussloch, Germany) at a
thickness of 40 ␮m. The sections were thaw-mounted onto Superfrost
Plus slides (Matsunami Glass, Osaka, Japan).
Immunohistochemical study. The immunohistochemical procedures were performed as described by Horie et al. (23) and Watanabe
et al. (38). Prior to staining, the slide-mounted sections were successively incubated in 10% normal donkey serum containing 0.2% Triton
X-100 and 0.1% sodium azide in PBS for 1 h, followed by PBS
containing 0.3% hydrogen peroxide for 30 min to quench endogenous
peroxidase activity, and were then washed three times for 10 min each
with PBS. In addition, the avidin and biotin sites of sections were
successively blocked using an avidin-biotin blocking kit (Vector
Laboratories, Peterborough, UK), and the sections were then washed
three times for 10 min each in PBS again. Subsequently, sections were
incubated in rabbit polyclonal anti-TRPV1 antibody for 40 h at room
temperature. Concentrations of the TRPV1 antibody (mouse TRPV1
C-terminus; Neuromics, Minneapolis, MN) were 1:60,000 for rectum,
1:40,000 for distal colon, and 1:20,000 for transverse and proximal
colon (Figs. 1, 4, and 5). To investigate the difference of the density
of TRPV1-immunoreactive nerve fibers in the rectum and distal,
transverse, and proximal colon, two different anti-TRPV1 antibodies
(1:60,000, Neuromics, and rat TRPV1 COOH-terminus, 1:1,000;
Trans Genic, Kumamoto, Japan) were used as the primary antibody
(Figs. 2 and 3). For immunoadsorption experiments, the antibody
(1:60,000; Neuromics) was preincubated with 10 ␮M of the corresponding antigen peptide (Neuromics) fragment for 1 h at room
temperature. After washes in PBS, the sections were incubated with
biotinylated donkey anti-rabbit immunoglobulin G (1:400; Jackson
Immunoresearch Laboratories, West Grove, PA) for 90 min at room
temperature. After further washes, the sections were incubated in
streptavidin biotin-peroxidase complex (1:5; Vectastain Elite ABC
kit, Vector Laboratories) for 1 h at room temperature, followed by
fluorescein isothiocyanate (FITC) tyramide (1:75; TSA kit, PerkinElmer Life Sciences, Boston, MA) (1). In control experiments, the
TRPV1 antibody was omitted from the staining procedures to verify
the specificity of the staining. No immunolabeling was observed in
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Male ddY-strain mice (Japan SLC, Hamamatsu, Japan) 6 – 8 wk old
were used. Animals were housed in a temperature-controlled room at
24°C with lights on from 0700 to 1900 and had free access to food and
water. All experiments were performed in compliance with the “Guiding Principles for the Care and Use of Laboratory Animals” approved
by the Japanese Pharmacological Society and the guidelines approved
by the institutional Animal Care and Use Committee of Josai International University. The protocols used were approved by the committee of Josai International University in accordance with the guidelines of APS guiding principles in the care and use of animals. The
number of animals used was kept to the minimum necessary for a
meaningful interpretation of the data, and animal discomfort was kept
to the minimum.
G349
G350
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
these controls. Double staining of the sections with TRPV1 antiserum
combined with a rabbit antiserum to the pan axonal marker protein
gene product (PGP) 9.5 was also carried out. In the case of PGP 9.5,
TRPV1 staining was carried out first by the TSA procedure described
above with FITC as the label and then followed by PGP 9.5 staining
(1:60,000; Biogenesis, Poole, UK) by the indirect labeling procedure.
To visualize the PGP 9.5 labeling, sections were then incubated for
4 h with donkey anti-rabbit secondary antibody linked to tetramethyl
rhodamine isothiocyanate (TRITC, 1:400; Jackson Immunoresearch
Laboratories). There was no cross-reactivity between TRPV1 and
PGP 9.5 staining.
Microscopy and image analysis. The sections were viewed at ⫻10
magnification (Zeiss Plan-NeoFluar) via an inverted fluorescence
microscope (Axioskop2 plus, Zeiss, Göttingen, Germany) equipped
with a filter for detection of fluorescein (FITC, 488 nm; TRITC; 543
nm). Randomly chosen images were acquired via a charge-coupled
device camera (AxioCam MRm, Zeiss), stored in a personal computer, and analyzed with Zeiss imaging software (Axiovision LE
version 4.6). To quantify TRPV1-positive axons, we measured
TRPV1-immunopositive areas in the image using the Automeasure
module (AutoMeasure Plus, Zeiss). Random fields were selected for
each slide. Next, thresholds for the brightness that represents the
TRPV1 immunoreactivity were selected by using the distribution of
brightness information. The TRPV1-positive area was calculated by
subtracting a nonspecific signal area of the control image (i.e., not
exposed to the antibody) from that of the experimental image (i.e.,
antibody treated) on serial cryostat sections of rectum or distal,
transverse, or proximal colon. In this manner, the absolute area of
TRPV1-positive axons could be determined for individual experAJP-Gastrointest Liver Physiol • VOL
iments and calculated as follows: TRPV1-positive area (%) ⫽
[(TRPV1 positive area/total area of antibody-treated section) ⫺
(positive area of control section/total area of the control section)] ⫻
100 (%).
For observation of the double staining of TRPV1 and PGP 9.5 in
the submucosal layer and myenteric plexus, the sections were viewed
at ⫻20 magnification (Olympus UPlanSApo) via a confocal microscope (FV-1000, Olympus, Tokyo, Japan). Multiple images in Zstacks were projected onto a single plane.
Statistical analysis. The data are expressed as means ⫾ SE.
Statistical analyses were performed by the two-tailed Student’s t-test
for comparison of two groups, and by a one-way analysis of variance
followed by a Bonferroni multiple-comparison test for comparison of
more than two groups. A P value ⬍ 0.05 was considered statistically
significant.
RESULTS
Localization and quantification of TRPV1 immunoreactivity
in isolated mouse lower gastrointestinal tract. Numerous
TRPV1-immunoreactive nerve fibers were seen in the mucosa,
submucosal layer, and myenteric plexus of the rectum and
distal colon (Fig. 1, A, B, and D). TRPV1-immunoreactive
fibers were also observed in the muscle layer and around
bundles of arterioles, venules, and lymphatic vessels in the
submucosal layer (Fig. 1C). TRPV1-immunoreactive fibers
were not observed after preincubation of the antibody with the
antigen peptide (Fig. 1E).
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 1. Distribution of transient receptor potential vanilloid type 1 (TRPV1) immunoreactivity in transverse
section of mouse rectum (A, C, D, and E) and distal
colon (B). MU, mucosa; CM, circular muscle. Numerous TRPV1-immunoreactive nerve fibers are found in
the mucosa (arrowheads), submucosal layer (concave
arrowheads), myenteric plexus (arrows), and mucosal
layer (double arrows). TRPV1-immunoreactive fibers
are also found around bundles of arterioles, venules, and
lymphatic vessels in the submucosal layer (C). Preadsorption of the primary antibody with the antigen peptide resulted in the loss of the signal (D and E). Scale
bar is 100 ␮m.
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
G351
The difference in the distributions of TRPV1 in mouse large
intestine was examined by using serial cryostat sections of the
rectum and distal, transverse, and proximal colon under the same
immunostaining condition (Fig. 2). To measure the amount of
TRPV1 immunoreactivity, two different anti-TRPV1 antibodies (Neuromics and Trans Genic) were used as the primary
antibody. In the rectum, numerous TRPV1-immunoreactive
nerve fibers were found in the mucosa, submucosal layer, and
muscle layer and myenteric plexus compared with control
tissues (Fig. 2, A and B). In the distal colon, the density of
TRPV1-positive nerve fibers was lower than in the rectum, but
they were clearly observed in the submucosal layer and myenteric plexus (Fig. 2, D and E). In contrast, the TRPV1positive axons were sparsely distributed in the transverse and
proximal colon (Fig. 2, G, H, J, K). Thus similar results were
obtained from both experiments using two different antiTRPV1 antibodies. In control experiments without the corresponding primary antibody, no immunolabeling was observed
(Fig. 2, C, F, I, and L).
To better understand the difference between the amounts of
TRPV1-immunoreactive nerve fibers in the rectum and distal,
transverse, and proximal colon, TRPV1-immunoreactive areas
were quantified by computerized binary image analysis (Fig.
3). Figure 3 shows TRPV1-immunoreactive areas in all layers
of the transverse section (Fig. 3A) and in the muscle layer
including myenteric plexus (Fig. 3B) using a TRPV1 antibody
AJP-Gastrointest Liver Physiol • VOL
(Neuromics). The TRPV1-immunopositive area in the rectum
was the largest in the isolated mouse lower gastrointestinal
tract. Both the rectum and distal colon contained a significantly
higher density of TRPV1-immunoreactive nerve fibers than the
transverse and proximal colon. The density of TRPV1-positive
nerve fibers was very low in the transverse and proximal colon.
These results were further confirmed by an immunohistochemical experiment using another TRPV1 antibody (Trans Genic,
Fig. 3, C and D).
Colocalization of TRPV1 with PGP 9.5. To investigate the
precise localization of TRPV1, we performed double-labeling
experiments with PGP 9.5, a pan axonal marker, to identify
neuronal cell bodies and nerve fibers using the fluorescence
microscope (Fig. 4) and confocal microscope (Fig. 5). Abundant TRPV1 immunoreactivity was detected in the myenteric
and submucosal plexuses of each part of the lower gastrointestinal tract (Fig. 4, A, D, G, and J). In all cases, the
immunoreactivity appeared axonlike in nature. TRPV1 staining of ganglionic cells was not detected in either submucosal or
myenteric plexuses in the rectum. PGP 9.5 staining was widely
distributed within the mucosa, submucosa, and smooth muscle
layer of each part of the lower gastrointestinal tract (Fig. 4, B,
E, H, and K) and did not show a clear difference among the
parts. Abundant PGP 9.5-immunoreactive fibers and bundles
were found in the myenteric plexus and submucosal layer, and
TRPV1 immunoreactivity profiles in these areas showed dou297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 2. Comparison of immunoreactivities to 2 different anti-TRPV1 antibodies in mouse rectum and
distal, transverse, and proximal colon. Left columns
(A, D, G, J) show TRPV1 labeling with anti-TRPV1
antibody (Neuromics). Middle columns (B, E, H, K)
show TRPV1 labeling with anti-TRPV1 antibody
(Trans Genic). Right columns (C, F, I, L) show the
control without the primary antibody. Scale bar is
100 ␮m.
G352
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
ble labeling with PGP 9.5 (Fig. 4, C, F, I, L). Figure 5 shows
the high-power magnification confocal images of the submucosal layer and myenteric plexus of rectum. Many TRPV1/
PGP 9.5 double-labeled axons were observed, but PGP 9.5immunoreactive cell bodies did not colocalize with TRPV1.
Difference in capsaicin-induced contractions in isolated
mouse rectum, distal, transverse, and proximal colon. Next, we
investigated the effect of the contraction induced by the
TRPV1 agonist capsaicin on smooth muscle tension in the
isolated mouse lower gastrointestinal tract. Figure 6 shows
typical recordings and concentration-response curves of the
capsaicin-induced contraction in mouse rectum and distal,
transverse, and proximal colon. In the rectum and distal colon,
capsaicin induced fast transient contractions, followed by
slowly developing long-lasting contractions that peaked within
2–3 min after administration (Fig. 6, A and B). The contractions induced by capsaicin were concentration dependent.
However, only transient contractions were observed in the
transverse and proximal colon (Fig. 6, C and D). No statistical
differences were found between 1 ␮M capsaicin-induced transient contractions in each part of the lower gastrointestinal
tract. Preexposure of tissues to capsaicin caused a dose-dependent desensitization to the second application of capsaicin in all
parts of the isolated lower gastrointestinal tract.
Effects of TTX, atropine, and TRPV1 antagonist BCTC on
capsaicin-induced contraction in isolated mouse lower gastrointestinal tract. To clarify the mechanism underlying contractile responses to TRPV1 activation by capsaicin in the mouse
rectum and distal, transverse, and proximal colon, we evaluated the effects of the neurotransmission blocker TTX, musAJP-Gastrointest Liver Physiol • VOL
carinic receptor antagonist atropine, and TRPV1 antagonist
BCTC on those contractile responses (Table 1). TTX abolished
transient contractile responses to capsaicin from the rectum to
the proximal colon, compared with each control group. Capsaicin-induced long-lasting contractions were significantly but
partially inhibited by TTX in the rectum and distal colon.
Atropine abolished the transient contraction from the rectum to
the proximal colon. The long-lasting contractions were significantly but partially inhibited by atropine in the rectum and
distal colon. The TRPV1 antagonist BCTC almost completely
inhibited both transient and long-lasting contractions in all
parts of the isolated mouse lower gastrointestinal tract. BCTC
(1 ␮M) did not affect the direct smooth muscle contractions in
response to 10 ␮M of acetylcholine (data not shown), but the
long-lasting contraction was not abolished by BCTC. Then we
performed a preliminary experiment with another TRPV1 antagonist, iodoresiniferatoxin, but it also failed to abolish the
capsaicin-induced long-lasting contraction (data not shown).
To investigate the capsaicin-induced relaxation in the mouse
colon, inhibition by nitric oxide synthase was examined. The
distal colon segments were preincubated with the nitric oxidesynthase inhibitor L-NAME (100 ␮M). L-NAME did not affect the
capsaicin-induced transient (control 33.1 ⫾ 3.4, L-NAME
31.34 ⫾ 12.6; n ⫽ 3– 6) or long-lasting contractions in the distal
colon (control 38.0 ⫾ 2.7, L-NAME 33.6 ⫾ 4.2; n ⫽ 3– 6).
Effects of tachykinin-receptor antagonists on capsaicin-induced contraction in isolated mouse lower gastrointestinal
tract. Tachykinins play an important role in the contractile
effect of capsaicin: they stimulate myenteric neurons and
mediate the neurogenic excitatory effects of capsaicin. We
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 3. Histogram showing positive areas (%)
of TRPV1 fibers in all layers (A and C), and in
the muscle layer including the myenteric
plexus (B and D) of the rectum and distal,
transverse, and proximal colon. Two kinds of
rabbit polyclonal anti-TRPV1 antibody (Neuromics; A and C) and (Trans Genic; B and
D) were used in each part of lower gastrointestinal tract. Each value represents means ⫾
SE of data obtained from 4 mice. Significantly different from the data of the rectum by
Bonferroni correction: **P ⬍ 0.01. #Significantly different from the data of the distal
colon by Bonferroni correction: #P ⬍ 0.05,
##P ⬍ 0.01.
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
G353
investigated the contractile effects of the tachykinins substance
P and neurokinin A on capsaicin-induced contraction in the
isolated mouse lower gastrointestinal tract (Fig. 7). Preliminary
experiments revealed that substance P (10 nM–1 ␮M) and
neurokinin A (10 nM–3 ␮M) induced dose-dependent contractions in the isolated mouse lower gastrointestinal tract. Therefore, we investigated the contractile response to substance P
and neurokinin A in the mouse rectum and distal, transverse,
and proximal colon (Fig. 7, A–D). The concentrations of
substance P and neurokinin A were chosen to induce submaximal contractions in each part. Substance P produced fast
transient contractions in all parts of the mouse isolated lower
gastrointestinal tract. Neurokinin A produced large long-lasting contractions in the rectum and distal colon that peaked 2–3
min after administration. In the transverse and proximal colon,
neurokinin A induced fast transient contractions, followed by
small long-lasting contractions. To determine the contribution
of the cholinergic pathways, responses to substance P and
neurokinin A were compared in the presence and absence of
the muscarinic receptor antagonist atropine (Fig. 7, A–D).
Atropine partially and significantly inhibited the substance
P-induced contractions, but no significant effect on the neurokinin A-induced contractions was observed in any part of the
mouse lower gastrointestinal tract.
Fig. 5. Confocal images showing TRPV1 and protein gene product (PGP) 9.5 double labeling in the
submucosal layer (A–C) and myenteric plexus
(D–F) of the mouse rectum. Sections were double
labeled with TRPV1 (green) and PGP 9.5 (red). The
TRPV1 and PGP 9.5 labelings are shown separately
in (A and D) and (B and E), respectively, and merged
(C and F). Arrowheads and arrows indicate the
colocalization of TRPV1/PGP 9.5 double-labeled
axons in the submucosal layer and myenteric plexus,
respectively. Scale bar is 50 ␮m.
AJP-Gastrointest Liver Physiol • VOL
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 4. Colocalization of TRPV1-positive nerve fibers with protein gene product (PGP) 9.5 in mouse
rectum and distal, transverse, and proximal colon.
Mouse lower gastrointestinal sections were double
labeled with TRPV1 (green) and PGP 9.5 (red). The
TRPV1 and PGP 9.5 labelings are shown separately
(A, D, G, J) and (B, E, H, K), respectively, and
merged (C, F, I, L). TRPV1 axons are sparse in the
smooth muscle layer but prominent in the myenteric
nerve plexus (arrows) and submucosal layer (concave arrowheads). In contrast, PGP 9.5 immunoreactive axons are abundant in all regions. Arrows and
concave arrowheads indicate the colocalization of
TRPV1 immunoreactivity with PGP 9.5 immunoreactivity. Scale bars are 100 ␮m.
G354
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 6. Typical recordings and concentrationresponse curves of capsaicin-induced contraction and desensitization in mouse rectum (A)
and distal (B), transverse (C), and proximal
colon (D). Typical recordings show that capsaicin induced a transient contraction, followed
by a long-lasting contraction in the rectum and
distal colon. In the transverse and proximal
colon, only the transient contraction was observed. Data for each contraction are expressed
as a percentage of the maximal contraction
induced by 10 ␮M acetylcholine (% of ACh
response) in each part of the lower gastrointestinal tract. Each value represents means ⫾ SE
of data obtained from 3–7 mice. No statistical
differences were found on 1 ␮M capsaicininduced transient contraction among each part
of lower gastrointestinal tract.
Next, we evaluated the effects of the NK1 receptor antagonist FK888 and the NK2 receptor antagonist GR159897 on
capsaicin-induced contractile responses (Fig. 8). Capsaicininduced transient contractions from the rectum to the proximal
colon were significantly and moderately inhibited by FK888 or
GR159897 compared with control responses (Fig. 8, A–D).
The combined blockade of NK1 and NK2 receptors with
FK888 and GR159897 markedly inhibited transient contractile
responses to capsaicin from the rectum to the proximal colon.
The long-lasting contractions in the rectum and distal colon
AJP-Gastrointest Liver Physiol • VOL
were significantly and moderately inhibited by GR159897, but
not by FK888 (Fig. 8, A and B). Coadministration of FK888
and GR159897 markedly inhibited long-lasting contractile responses to capsaicin in the rectum and distal colon.
The substance P (100 nM)-induced contractions in the rectum and distal, transverse, and proximal colon were significantly blocked by 10 ␮M FK888 (rectum: control 59.2 ⫾ 7.9,
FK888 8.4 ⫾ 1.7, P ⬍ 0.01; distal colon: control 66.6 ⫾ 11.6,
FK888 18.2 ⫾ 3.8, P ⬍ 0.05; transverse colon: control 61.3 ⫾
11.3, FK888 20.5 ⫾ 3.4, P ⬍ 0.05; proximal colon: control
297 • AUGUST 2009 •
www.ajpgi.org
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
Table 1. Effect of TTX, atropine, or TRPV1 antagonist
BCTC on capsaicin-induced contraction in isolated mouse
rectum, distal colon, transverse colon, and proximal colon
Control
Rectum
Transient contraction
Long-lasting
contraction
Distal colon
Transient contraction
Long-lasting
contraction
Transverse colon
Transient contraction
Proximal colon
Transient contraction
TTX, 300 nM Atropine, 1 M BCTC, 1 M
33.2⫾5.5
1.6⫾1.6†
1.3⫾1.3†
3.1⫾0.6†
50.4⫾3.4
36.7⫾3.5*
35.8⫾5.4*
7.0⫾0.3†
33.1⫾3.4
0.0⫾0.0†
0.5⫾0.3†
3.7⫾1.2†
38.6⫾2.7
25.4⫾3.3*
14.3⫾4.6†
5.7⫾1.9†
22.3⫾3.7
0.0⫾0.0†
3.7⫾2.2†
1.6⫾0.3†
30.0⫾5.5
0.0⫾0.0†
6.4⫾4.2*
1.5⫾0.3†
60.8 ⫾ 8.2, FK888 18.4 ⫾ 3.8%, P ⬍ 0.01; n ⫽ 5– 8), but not
significantly by 3 ␮M GR159897 (rectum: control 59.2 ⫾ 7.9,
GR159897 49.9 ⫾ 8.5; distal colon: control 66.6 ⫾ 11.6,
GR159897 61.9 ⫾ 12.6; transverse colon: control 61.3 ⫾
11.3, GR159897 53.8 ⫾ 10.4; proximal colon: control 60.8 ⫾
8.2, GR159897 62.4 ⫾ 10.8%; n ⫽ 5– 8). The neurokinin A
(300 nM or 1 ␮M)-induced contractions in the rectum and
distal, transverse, and proximal colon were significantly
blocked by 3 ␮M GR159897 (rectum: control 72.7 ⫾ 15.9,
GR159897, 9.4 ⫾ 2.6, P ⬍ 0.05; distal colon: control 65.3 ⫾
16.5, GR159897 18.4 ⫾ 4.5, P ⬍ 0.05; transverse colon:
control 46.0 ⫾ 8.5, GR159897 9.0 ⫾ 2.7, P ⬍ 0.01; proximal
colon: control 31.2 ⫾ 4.1, GR159897 6.8 ⫾ 1.6%, P ⬍ 0.01;
n ⫽ 5– 8), but not significantly by 10 ␮M FK888 (rectum:
control 72.7 ⫾ 15.9, FK888, 81.1 ⫾ 13.8; distal colon: control 65.3 ⫾ 16.5, FK888 79.3 ⫾ 19.3; transverse colon: control
46.0 ⫾ 8.5, FK888 47.7 ⫾ 18.7; proximal colon: control
31.2 ⫾ 4.1, FK888 36.4 ⫾ 8.4%; n ⫽ 5– 8). An NK3 receptor
blockade partially inhibited the contraction induced by capsaicin, suggesting the involvement of NK3 receptors in the
response to capsaicin in mouse jejunum (14). We tried to
clarify this by using the commercially available NK3 antagonist SB222200 (30). SB222200 (30 ␮M) inhibited the neurokinin B-induced contraction, but it also markedly inhibited the
substance P- and neurokinin A-induced contractions. Therefore, the involvement of NK3 receptors in capsaicin-induced
contraction is unclear at this time (data not shown).
DISCUSSION
This study was designed to investigate differences in the
distribution of TRPV1 channels and the effects of capsaicin on
smooth muscle contraction using isolated mouse lower gastrointestinal tract segments. The localization of TRPV1 channels
and their effects on motor function in the large intestine,
especially site differences, remain to be elucidated. In the
present study, we isolated the mouse rectum and distal, transverse, and proximal colon and examined the pharmacological
and physiological differences among the parts by using immunohistochemical methods and tension recordings of capsaicininduced contractions. The present results suggest that TRPV1
AJP-Gastrointest Liver Physiol • VOL
channels located on the rectum and distal colon play a major
role in the motor function of the mouse lower gastrointestinal
tract. Furthermore, neurokinin A was found to be the main
neurotransmitter in large long-lasting capsaicin-induced contractions in the mouse rectum and distal colon.
First, we mapped the localization and distribution of TRPV1
in the mouse lower gastrointestinal tract using immunofluorescence. To increase the sensitivity of immunodetection, we
visualized anti-TRPV1 antibodies using an amplification
method based on the catalyzed deposition of fluoresceinconjugated tyramide (1). In this study, we demonstrate TRPV1
immunoreactivity in the mucosa, submucosal layer, muscle
layer, and myenteric plexus of mouse lower gastrointestinal
tract. In the mucosal layer of the rectum and distal colon,
TRPV1-immunoreactive axons were observed running across
and along the mucus glands. In the submucosal layer, TRPV1immunoreactive fibers were found around bundles of arterioles, venules, and lymphatic vessels. In our previous study of
rat stomach, TRPV1 axons were observed running along gastric glands in the mucosa and around blood vessels in the
submucosal layer, suggesting the role of TRPV1 nerve fibers in
gastric acid and mucus secretion (22, 23). The gastrointestinal
protection produced by capsaicin-induced stimulation of sensory neurons is associated with a marked increase of mucosal
blood flow in the small and large intestine of experimental
animals (18). These findings suggest that the TRPV1 nerve
fibers of the mucosal layer are involved in mucus secretion and
blood flow in the mouse large intestine. In the muscle layer,
TRPV1-immunoreactive fibers were observed in the rectal side
of the mouse lower gastrointestinal tract.
Extrinsic primary afferent neurons have their cell bodies in
nodose and jugular ganglia or dorsal root ganglia. TRPV1positive nerve fibers in the gut appear to originate predominantly in spinal afferents, with very few in vagal afferents (37).
Other investigators reported that TRPV1 immunoreactivity
was detected on both intrinsic and extrinsic neurons in the gut
(3), and TRPV1 mRNA has also been reported in the mouse
colon (25, 32). Ward et al. (37) reported that TRPV1 nerve
fibers form a network around myenteric nerve cell bodies in the
mouse, guinea pig, and rat gastric antrum. Consistent with
previous findings for TRPV1/PGP 9.5 double staining (23, 37)
in the gastrointestinal tract, we observed TRPV1/PGP 9.5
double-labeled axons in the myenteric plexus. However, PGP
9.5-immunoreactive cell bodies were not colocalized with
TRPV1 immunoreactivity. Pharmacological studies have shown that
the population of capsaicin-sensitive gastrointestinal neurons is
composed of extrinsic primary afferents, whereas intrinsic
enteric neurons do not respond directly to capsaicin (8). Taken
together, the data imply that TRPV1-expressing nerve fibers
observed in the myenteric plexus are extrinsic primary afferents, but further studies are needed to determine the origin of
the TRPV1 channels in the mouse large intestine.
The difference between the densities of TRPV-immunoreactive nerve fibers in the rectum and distal, transverse, and
proximal colon has not been studied under physiological conditions. In the present study, the TRPV1-immunoreactive areas
of each segment were quantified by computerized binary image
analysis using two anti-TRPV1 antibodies. Interestingly, high
densities of TRPV1-positive areas were detected in the rectum
and distal colon, but not in the transverse and proximal colon.
In contrast, clear differences in PGP 9.5 densities were not
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Each value represents the mean ⫾ SE of 5–7 animals. TTX, tetrodotoxin;
BCTC, N-(4-tertiary butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine1(2H)-carbox-amide. Values that were significantly different from those of the
control group: *P ⬍ 0.05, †P ⬍ 0.01.
G355
G356
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
AJP-Gastrointest Liver Physiol • VOL
297 • AUGUST 2009 •
www.ajpgi.org
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
of capsaicin is attributed to a direct activation of smooth
muscle stimulated by neurotransmitters from sensory nerve
endings (14). In the present study, capsaicin-induced transient
contractions in the rectum to proximal colon were abolished by
TTX and atropine. The long-lasting contractions in the rectum
and distal colon were partially inhibited by TTX and atropine.
The amplitudes of transient and long-lasting contractions were
reduced by repeated administration of capsaicin, which is one
peculiar effect of TRPV1 activation. To study the involvement
of TRPV1 in isolated gastrointestinal preparations, we used
BCTC as a selective TRPV1 antagonist (33). BCTC almost
completely inhibited the response to capsaicin in all gastrointestinal segments without affecting the direct smooth muscle
contraction, as previously reported for isolated mouse jejunum
(14). However, we observed that the long-lasting contraction
was not abolished even with adequate BCTC treatment. Next,
we performed a preliminary experiment with another TRPV1
antagonist, iodoresiniferatoxin, but iodoresiniferatoxin also
failed to abolish the capsaicin-induced long-lasting contraction. Therefore, capsaicin is thought to elicit the long-lasting
contraction partly via the TRPV1-independent pathway. De
Man et al. (14) also reported that TRPV1 antagonist iodoresiniferatoxin partially inhibited the contraction to capsaicin in
mouse jejunum.
Benko et al. (10) reported that capsaicin induces nitric
oxide-mediated relaxation in circular muscle-oriented preparations isolated from human and mouse colon. In the present
study, we measured the longitudinal tension in longitudinally
oriented muscle preparations. Pretreatment with L-NAME did
not affect the capsaicin-induced contraction in the mouse distal
colon, suggesting that nitric oxide is not involved in this
response. Furthermore, in our preliminary experiments, we
never observed nitric oxide-mediated relaxation in colonic and
rectal longitudinal muscle treated with capsaicin when the
isolated segments were preincubated with the NK1 antagonist
FK888, the NK2 antagonist GR159897, or atropine, unlike the
report of Benko et al. It is thought that the difference between
Benko’s and our results arises from the preparations used.
Taken together, these results suggest that the capsaicininduced transient contraction is mediated by intrinsic cholinergic activation, followed by the release of sensory neurotransmitters from the extrinsic sensory nerve. The long-lasting
contraction is mediated mainly by neurotransmitters released
from extrinsic sensory nerve endings and partially by the
cholinergic pathway in the mouse lower gastrointestinal tract.
It is well known that tachykinins released from capsaicinsensitive primary afferents elicit contractions of smooth muscle
by a local efferent action (8). The involvement of tachykinin
NK1 and NK2 receptors, which prefer substance P and neurokinin A, respectively, in the modulation of capsaicin-induced
contractions has been shown in the guinea pig ileum. The
distributions of substance P, neurokinin A, NK1 receptors, and
NK2 receptors have been studied by immunohistochemical
Fig. 7. Typical recordings showing contractile response to substance P and neurokinin A in mouse rectum (A) and distal (B), transverse (C), and proximal colon
(D). The concentrations of substance P and neurokinin A used in the rectum and distal, transverse, and proximal colon were submaximal concentrations that were
determined by concentration response curves to substance P and neurokinin A in each segment. The effects of atropine (1 ␮M) on the contractile effects of
substance P and neurokinin A in mouse rectum to proximal colon. The concentrations of substance P and neurokinin A used in each segment were that same
as shown in each typical recording. Data are expressed as a percentage of the maximal contraction induced by 10 ␮M acetylcholine in each part of the lower
gastrointestinal tract. Each value represents means ⫾ SE of data obtained from 5– 8 mice. *Significantly different from those of the control by Student’s t-test
(*P ⬍ 0.05, **P ⬍ 0.01).
AJP-Gastrointest Liver Physiol • VOL
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
observed from the proximal colon to the rectum. Consistent
with the binary image analysis of the muscle layers including
the myenteric plexus in each segment, the capsaicin-induced
contraction patterns were different on the anal (rectum and
distal colon) and oral (transverse and proximal colon) sides of
the mouse large intestine. These contraction patterns were
characterized by transient and subsequent long-lasting contractions in the rectum and distal colon and only transient contractions in the transverse and proximal colon. Thus we speculated
that TRPV1 channels located on the rectum and distal colon
play a major role in the motor function. On the basis of our in
vitro data, we speculated that capsaicin stimulates colonic
motility in the in vivo experiment. Indeed, it is reported that
intracolonic administration of capsaicin increases colonic motility in a dose-dependent manner in conscious dogs (17).
Therefore, the activation of TRPV1 channels is thought to
result in the promotion of motility in the colon.
TRPV1 channels located in the mucosa are considered to
play important roles in detecting visceral pain because TRPV1
contributes to the perception of noxious mechanical stimuli and
inflammatory stimuli in the mouse colon (24, 29). In the
gastrointestinal tract, TRPV1 channels play both protective and
nonprotective roles against inflammatory colitis (35). Holzer
(20) suggested that TRPV1 channels involved in gastrointestinal protection may be differentiated pharmacologically from
TRPV1 channels mediating gastrointestinal inflammation. Azuma
et al. (6) reported that the histological colitis score of the distal
colon was higher than that of the proximal colon in dextran
sodium sulfate-induced colitis. In this study, we clearly demonstrated that the TRPV1 channel density in the mucosal area
is greater in the rectum and distal colon than in the transverse
and proximal colon. Thus the abundant TRPV1 channel expression may result in the induction of more pronounced colitis
in the rectum and distal colon than in the transverse and
proximal colon. However, further studies are needed to clarify
the roles of TRPV1 channels in colitis.
In the guinea pig esophagus, capsaicin induces a biphasic
contraction, i.e., immediate transient and late long-lasting contractions (9). A similar contraction pattern was observed in the
mouse rectum and distal colon in the present study. The
contraction due to capsaicin is well studied in guinea pig ileum,
and it is known that biologically active substances released
from sensory nerves stimulate both smooth muscle and intrinsic myenteric neurons (8). Acetylcholine released from intrinsic cholinergic motor neurons is one of the final mediators of
the capsaicin-induced contraction. Capsaicin-sensitive sensory
nerves innervating the gastrointestinal tract are known to be
TTX resistant (5, 11). In the guinea pig ileum, capsaicin
induces TTX-sensitive acetylcholine release, suggesting that
the TTX- and atropine-sensitive contractile effects of capsaicin
are based on acetylcholine release from myenteric cholinergic
neurons stimulated by neurotransmitters from sensory nerves
(34). On the other hand, the TTX-insensitive contractile effect
G357
G358
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig. 8. Effects of NK1 antagonist FK888 (10
␮M), NK2 antagonist GR159897 (3 ␮M), and
their combination on capsaicin-induced transient and long-lasting contractions in mouse
rectum (A) and distal (B), transverse (C), and
proximal colon (D). Data of each contraction
are expressed as a percentage of the maximal
contraction induced by 10 ␮M acetylcholine
in each part of the lower gastrointestinal tract.
Each value represents means ⫾ SE of data
obtained from 5– 8 mice. *Significantly different from the control by Bonferroni test
(*P ⬍ 0.05, **P ⬍ 0.01).
methods. Substance P and neurokinin A are expressed in
extrinsic and intrinsic primary afferent neurons and myenteric
neurons (interneurons, excitatory motor neurons) (26). NK1
receptor immunoreactivity has been found in the myenteric
plexus and muscle layer (26, 36). The NK2 receptors are found
in longitudinal and circular muscle fibers of the mouse ileum
and are thought to be expressed mainly on the smooth muscle
layer (26, 31). To investigate the pattern of contraction induced
by the NK1 and NK2 receptor activation, we compared exogeAJP-Gastrointest Liver Physiol • VOL
nous substance P- and neurokinin A-induced contractions,
respectively. We observed atropine-sensitive, fast transient
contractions induced by substance P in all parts of the isolated
mouse lower gastrointestinal tract. Neurokinin A produced
large atropine-insensitive, long-lasting contractions in the rectum and distal colon. In the transverse and proximal colon,
neurokinin A induced fast transient contractions, followed by
small long-lasting contractions. The reactivities to neurokinin
A of the rectum and distal colon were higher than those of the
297 • AUGUST 2009 •
www.ajpgi.org
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
ACKNOWLEDGMENTS
We thank Dr. Naoto Watanabe (Department of Respiratory and Infectious
Diseases, St. Marianna University School of Medicine; Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University)
for helpful advice and for discussions of the immunohistochemical study.
GRANTS
This work was supported in part by Grants-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports, and Culture of Japan (nos.
AJP-Gastrointest Liver Physiol • VOL
19590156, 18790126, 18790127, and 20790075) and by the Uehara Memorial
Foundation.
REFERENCES
1. Adams JC. Biotin amplification of biotin and horseradish peroxidase
signals in histochemical stains. J Histochem Cytochem 40: 1457–1463,
1992.
2. Akbar A, Yiangou Y, Facer P, Walters JR, Anand P, Ghosh S.
Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable
bowel syndrome and their correlation with abdominal pain. Gut 57:
923–929, 2008.
3. Anavi-Goffer S, Coutts AA. Cellular distribution of vanilloid VR1
receptor immunoreactivity in the guinea-pig myenteric plexus. Eur
J Pharmacol 458: 61–71, 2003.
4. Andrade EL, Ferreira J, André E, Calixto JB. Contractile mechanisms
coupled to TRPA1 receptor activation in rat urinary bladder. Biochem
Pharmacol 72: 104 –114, 2006.
5. Arbuckle JB, Docherty RJ. Expression of tetrodotoxin-resistant sodium
channels in capsaicin-sensitive dorsal root ganglion neurons of adult rats.
Neurosci Lett 185: 70 –73, 1995.
6. Azuma YT, Hagi K, Shintani N, Kuwamura M, Nakajima H, Hashimoto H, Baba A, Takeuchi T. PACAP provides colonic protection
against dextran sodium sulfate induced colitis. J Cell Physiol 216: 111–
119, 2008.
7. Barthó L, Benkó R, Lázár Z, Illényi L, Horváth OP. Nitric oxide is
involved in the relaxant effect of capsaicin in the human sigmoid colon
circular muscle. Naunyn Schmiedebergs Arch Pharmacol 366: 496 –500,
2002.
8. Barthó L, Benkó R, Patacchini R, Pethö G, Holzer-Petsche U, Holzer
P, Lázár Z, Undi S, Illényi L, Antal A, Horváth OP. Effects of capsaicin
on visceral smooth muscle: a valuable tool for sensory neurotransmitter
identification. Eur J Pharmacol 500: 143–157, 2004.
9. Barthó L, Lénárd L Jr, Patacchini R, Halmai V, Wilhelm M, Holzer
P, Maggi CA. Tachykinin receptors are involved in the “local efferent”
motor response to capsaicin in the guinea-pig small intestine and oesophagus. Neuroscience 90: 221–228, 1999.
10. Benko R, Lazar Z, Undi S, Illenyi L, Antal A, Horvath OP, Rumbus
Z, Wolf M, Maggi CA, Bartho L. Inhibition of nitric oxide synthesis
blocks the inhibitory response to capsaicin in intestinal circular muscle
preparations from different species. Life Sci 76: 2773–2782, 2005.
11. Campbell DT. Large and small vertebrate sensory neurons express
different Na and K channel subtypes. Proc Natl Acad Sci USA 89:
9569 –9573, 1992.
12. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD,
Julius D. The capsaicin receptor: a heat-activated ion channel in the pain
pathway. Nature 389: 816 – 824, 1997.
13. Chan CL, Facer P, Davis JB, Smith GD, Egerton J, Bountra C,
Williams NS, Anand P. Sensory fibres expressing capsaicin receptor
TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet
361: 385–391, 2003.
14. De Man JG, Boeckx S, Anguille S, de Winter BY, de Schepper HU,
Herman AG, Pelckmans PA. Functional study on TRPV1-mediated
signalling in the mouse small intestine: involvement of tachykinin receptors. Neurogastroenterol Motil 20: 546 –556, 2008.
15. Furness JB. Types of neurons in the enteric nervous system. J Auton Nerv
Syst 8: 87–96, 2000.
16. Furness JB, Jones C, Nurgali K, Clerc N. Intrinsic primary afferent
neurons and nerve circuits within the intestine. Prog Neurobiol 72:
143–164, 2004.
17. Hayashi K, Shibata C, Ueno T, Nagao M, Kudo K, Sato M, Sasaki I.
Intracolonic capsaicin stimulates colonic motility and defecation via cholinergic receptors in conscious dogs. Neurogastroenterol Motil 19: 70 –71,
2007.
18. Holzer P. Efferent-like roles of afferent neurons in the gut: blood flow
regulation and tissue protection. Auton Neurosci 125: 70 –75, 2006.
19. Holzer P. TRPV1 and the gut: from a tasty receptor for a painful vanilloid
to a key player in hyperalgesia. Eur J Pharmacol 500: 231–241, 2004.
20. Holzer P. Vanilloid receptor TRPV1: hot on the tongue and inflaming the
colon. Neurogastroenterol Motil 16: 697– 699, 2004.
21. Holzer P, Holzer-Petsche U. Tachykinin receptors in the gut: physiological and pathological implications. Curr Opin Pharmacol 1: 583–90, 2001.
22. Horie S, Michael GJ, Priestley JV. Co-localization of TRPV1-expressing nerve fibers with calcitonin-gene-related peptide and substance P in
fundus of rat stomach. Inflammopharmacology 13: 127–137, 2005.
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
transverse and proximal colon. It is suggested that NK1 and
NK2 receptor activation induces a capsaicin-like contractile
response in the mouse lower gastrointestinal tract.
Therefore, we next investigated the effects of the NK1
receptor and/or the NK2 receptor blockade on capsaicin-induced contractile responses. Our results show that the capsaicin-induced transient contraction was significantly inhibited
by NK1 or NK2 receptor antagonists, and a combined blockade
of NK1 and NK2 receptors abolished transient contractile
responses to capsaicin, suggesting that NK1 and NK2 receptors
cooperate in producing the transient contraction. On the other
hand, the long-lasting capsaicin-induced contraction was significantly inhibited by an NK2 antagonist but not by an NK1
antagonist, indicating that the release of neurokinin A from
sensory nerve endings is mainly responsible for the longlasting contraction. Altogether, these results indicate that different mechanisms are involved in the transient and longlasting contractions induced by capsaicin in the mouse lower
gastrointestinal tract, i.e., the intrinsic cholinergic and extrinsic
sensory pathways are the main pathways of transient and
long-lasting contractions, respectively. Substance P and neurokinin A released from sensory nerves stimulate NK1 and
NK2 receptors on parasympathetic ganglia, leading to acetylcholine release from cholinergic nerve endings. This is the
putative mechanism of capsaicin-induced transient contractions in the rectum and proximal colon. In the long-lasting
contraction in the rectum and distal colon, the major final
mediator is the neurokinin A released from extrinsic sensory
nerve endings, which activates NK2 receptors on the smooth
muscle cells. Our results suggest that TRPV1 channels located
on the rectal and distal colon play a major role in the motor
function of the large intestine. The differences between capsaicin-induced contractions in the rectum, distal colon, transverse
colon, and proximal colon segments may be attributed to these
differences in the TRPV1 density and neurokinin A reactivity.
Although the present study provides significant information
on the mechanisms of capsaicin-induced contractions in the
mouse lower gastrointestinal tract, the localization of endogenous tachykinins and tachykinin receptors remains unknown.
Colocalization studies on tachykinins and/or tachykinin receptors with TRPV1 channels may support the involvement of
tachykinins, especially neurokinin A and NK2 receptors, in the
mechanism of TRPV1-mediated motor function of the rectum
and distal colon.
The present results provide new information regarding the
site specificity of TRPV1 channels in the mouse lower gastrointestinal tract. Our findings suggest that TRPV1 channels
located on the rectum and distal colon play a major role in the
motor function of the large intestine. Further studies of TRPV1
channels in the rectum and distal colon may provide significant
information about the role of TRPV1 in regulating a wide
variety of functions in the large intestine.
G359
G360
LOCALIZATION AND MOTOR FUNCTION OF TRPV1 CHANNELS
AJP-Gastrointest Liver Physiol • VOL
31. Patterson LM, Zheng H, Ward SM, Berthoud HR. Vanilloid receptor
(VR1) expression in vagal afferent neurons innervating the gastrointestinal
tract. Cell Tissue Res 311: 277–287. 2003.
32. Penuelas A, Tashima K, Tsuchiya S, Matsumoto K, Nakamura T,
Horie S, Yano S. Contractile effect of TRPA1 receptor agonists in the
isolated mouse intestine. Eur J Pharmacol 576: 143–150, 2007.
33. Pomonis JD, Harrison JE, Mark L, Bristol DR, Valenzano KJ,
Walker K. N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: II. In vivo characterization in rat models of inflammatory and neuropathic pain. J Pharmacol Exp Ther 306: 387–393, 2003.
34. Someya A, Horie S, Yamamoto H, Murayama T. Modifications of
capsaicin-sensitive neurons in isolated guinea pig ileum by [6]-gingerol
and lafutidine. J Pharm Sci 92: 359 –366, 2003.
35. Storr M. TRPV1 in colitis: is it a good or a bad receptor?—a viewpoint.
Neurogastroenterol Motil 19: 625– 629, 2007.
36. Vannucchi MG, Faussone-Pellegrini MS. NK1, NK2 and NK3 tachykinin receptor localization and tachykinin distribution in the ileum of the
mouse. Anat Embryol (Berl) 202: 247–255, 2000.
37. Ward SM, Bayguinov J, Won KJ, Grundy D, Berthoud HR. Distribution of the vanilloid receptor (VR1) in the gastrointestinal tract. J Comp
Neurol 465: 121–135, 2003.
38. Watanabe N, Horie S, Michael GJ, Keir S, Spina D, Page CP, Priestley
JV. Immunohistochemical co-localization of transient receptor potential
vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory
system. Neuroscience 141: 1533–1543, 2006.
39. Yiangou Y, Facer P, Dyer NH, Chan CL, Knowles C, Williams NS,
Anand P. Vanilloid receptor 1 immunoreactivity in inflamed human
bowel. Lancet 357: 1338 –1339, 2001.
297 • AUGUST 2009 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.5 on June 18, 2017
23. Horie S, Yamamoto H, Michael GJ, Uchida M, Belai A, Watanabe K,
Priestley JV, Murayama T. Protective role of vanilloid receptor type 1 in
HCl-induced gastric mucosal lesions in rats. Scand J Gastroenterol 39:
303–312, 2004.
24. Jones RC, Xu L, Gebhart GF. The mechanosensitivity of mouse colon
afferent fibers and their sensitization by inflammatory mediators require
transient receptor potential vanilloid 1 and acid-sensing ion channel 3.
J Neurosci 25: 10981–10989, 2005.
25. Kimball ES, Prouty SP, Pavlick KP, Wallace NH, Schneider CR,
Hornby PJ. Stimulation of neuronal receptors, neuropeptides and cytokines during experimental oil of mustard colitis. Neurogastroenterol Motil
19: 390 – 400, 2007.
26. Lecci A, Capriati A, Maggi CA. Tachykinin NK2 receptor antagonists
for the treatment of irritable bowel syndrome. Br J Pharmacol 141:
1249 –1263, 2004.
27. Maggi CA, Giuliani S, Santicioli P, Patacchini R, Said SI, Theodorsson E, Turini D, Barbanti G, Giachetti A, Meli A. Direct evidence for
the involvement of vasoactive intestinal polypeptide in the motor response
of the human isolated ileum to capsaicin. Eur J Pharmacol 185: 169 –178,
1990.
28. Maggi CA, Theodorsson E, Santicioli P, Patacchini R, Barbanti G,
Turini D, Renzi D, Giachetti A. Motor response of the human isolated
colon to capsaicin and its relationship to release of vasoactive intestinal
polypeptide. Neuroscience 39: 833– 841, 1990.
29. Miranda A, Nordstrom E, Mannem A, Smith C, Banerjee B, Sengupta
JN. The role of transient receptor potential vanilloid 1 in mechanical and
chemical visceral hyperalgesia following experimental colitis. Neuroscience 148: 1021–1032, 2007.
30. Onori L, Aggio A, Taddei G, Ciccocioppo R, Severi C, Carnicelli V,
Tonini M. Contribution of NK3 tachykinin receptors to propulsion in the
rabbit isolated distal colon. Neurogastroenterol Motil 13: 211–219, 2001.