Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.
com
Gut Online First, published on February 21, 2014 as 10.1136/gutjnl-2013-305870
1
Division of Gastroenterology,
Laboratory of Experimental
Medicine and Pediatrics,
University of Antwerp,
Antwerp, Belgium
2
Department of
Gastroenterology, Antwerp
University Hospital, Antwerp,
Belgium
Correspondence to
Professor Benedicte De Winter,
Laboratory of Experimental
Medicine and Pediatrics,
Division of Gastroenterology,
University of Antwerp, Campus
Drie Eiken, Universiteitsplein 1,
Antwerp B-2610, Belgium;
benedicte.dewinter@
uantwerpen.be
Received 12 August 2013
Revised 21 January 2014
Accepted 28 January 2014
To cite: Deiteren A, De
Man JG, Ruyssers NE, et al.
Gut Published Online First:
[please include Day Month
Year] doi:10.1136/gutjnl-
2013-305870
Copyright
Deiteren A, etArticle author (or
al. Gut 2014;0:1–10.
doi:10.1136/gutjnl-2013-305870
Neurogastroenterology
ORIGINAL ARTICLE
Histamine H4 and H1 receptors contribute
to postinflammatory visceral hypersensitivity
Annemie Deiteren,1 Joris G De Man,1 Nathalie E Ruyssers,1 Tom G Moreels,1,2
Paul A Pelckmans,1,2 Benedicte Y De Winter1
ABSTRACT
Objectives Substantial evidence implicates mast cells
and their main constituent histamine in the pathogenesis
of visceral hypersensitivity. We explored the specific
contribution of histamine H4 (H4R) and H1 (H1R)
receptors to visceral hypersensitivity in a
postinflammatory rat model.
Design Trinitrobenzenesulfonic acid (TNBS)-colitis was
monitored individually by colonoscopy: first on day 3 to
confirm the presence of colitis and then every 4 days,
starting from day 10, to monitor convalescence and
determine the exact timepoint of endoscopic healing in
each rat. Experiments were performed 3 days after
endoscopic resolution of colitis. Visceral sensitivity was
assessed by quantifying visceromotor responses (VMRs)
to colorectal distension. Colonic mast cell numbers,
histamine release and H4R and H1R mRNA expression
were quantified. JNJ7777120 (H4R antagonist) and/or
levocetirizine (H1R antagonist) were administered 30 min
prior to VMR assessment or histamine release assay.
Results Postcolitis rats displayed a higher number of
colonic mast cells, excessive histamine release and
significantly enhanced VMRs. Heightened VMRs were
dose-dependently reduced by JNJ7777120 and
levocetirizine; combined administration of JNJ7777120
and levocetirizine potentiated the antinociceptive effect.
In the colon, both H4R and H1R mRNA were present; in
the dorsal root ganglia, only H1R mRNA was found.
Only colonic H4R mRNA expression was increased in
postcolitis rats. Excessive histamine release in postcolitis
rats was attenuated by the highest dose of JNJ7777120.
Conclusions H4R and H1R antagonists dose-
dependently reduce and even normalise
postinflammatory visceral hypersensitivity via different
underlying mechanisms but with a synergistic effect.
Both receptor subtypes represent promising targets for
the treatment of postinflammatory visceral
hypersensitivity.
INTRODUCTION
IBS is a functional gastrointestinal disorder charac-
terised by chronic abdominal pain and an altered
bowel habit. Although the exact pathophysiology is
yet to be elucidated, visceral hypersensitivity, dis-
turbed intestinal motor function and psychological
aspects have been identified as important contribu-
tors.1 Visceral hypersensitivity is considered the main
factor underlying abdominal pain in IBS and, indeed,
enhanced pain perception in response to colorectal
distension is present in the majority of IBS patients
and has been shown to correlate with abdominal pain
symptoms.1–3 However, treatment options for
their employer) 2014. Produced by BMJ Publishing Group Ltd (& BSG) under licence.
Significance of this study
What is already known on this subject?
▸ Mast cells and their main mediator histamine
contribute significantly to visceral
hypersensitivity.
▸ A recent clinical trial highlights the therapeutic
potential of H1 receptor (H1R) antagonists for
the treatment of IBS symptoms.
What are the new findings?
▸ Selective blockade of the H4R or H1R
dose-dependently reduced in vivo
postinflammatory visceral hypersensitivity in a
rat model, and there was a functional interplay
between both receptor subtypes.
▸ Only H1R mRNA was present in the dorsal root
ganglia, whereas both H4R and H1R mNRA
were present in the colon. Colonic H4R mRNA
expression was increased in the
postinflammation group.
How might it impact on clinical practice in
the foreseeable future?
▸ Our findings reveal a therapeutic potential of
selective H4R and H1R-targeted therapies,
alone or in combination, for postinflammatory
visceral hypersensitivity, supporting the
rationale for H1R-directed clinical trials and
uncovering a novel target, the H4R.
IBS-related visceral pain are still limited and patients
and physicians eagerly await novel therapeutic
strategies.4
A potential target in this quest for new therapies
is the mast cell (MC) and its main mediator hista-
mine. Several lines of evidence corroborate MC
involvement in visceral hypersensitivity.5–10 Both
the location of MCs in close apposition to afferent
nerves in the gut wall and the bidirectional commu-
nication between them both point towards a poten-
tial role for MCs in the sensitisation of afferent
nerves.5 6 Moreover, activated MCs in close prox-
imity to the colonic nerves were shown to correlate
with the severity and frequency of abdominal pain
in IBS patients.7 Functionally, mucosal MCs
obtained from IBS patients show a higher rate of
activation and release greater amounts of histamine
and tryptase.7 In addition, IBS colonic biopsy
1
 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Neurogastroenterology

supernatants, containing increased levels of these MC media-
tors, excite dorsal root ganglia (DRG) neurons, increase the
firing rate of mesenteric afferent nerves and induce visceral
hyperalgesia when applied in animal models.8–10 Moreover, the
excitatory action of IBS supernatant on guinea pig enteric
neurons was more pronounced for hypersensitive compared
with normosensitive patients and correlated with patients’
degree of visceral hypersensitivity.11
Histamine is the main MC mediator and binds to four receptor
subtypes (H1-4) of which H1 and H4 may be interesting targets
for visceral pain. Recently, ketotifen, a MC stabiliser with H1
receptor (H1R) antagonistic properties, increased the threshold of
discomfort to rectal distension in IBS patients with documented
visceral hypersensitivity and improved IBS symptoms such as
abdominal pain.12 Interestingly, ketotifen did not affect MC
numbers or their mediator release, leading the authors to suggest
that the beneficial effect of ketotifen was mediated by its H1R
antagonistic properties rather than by stabilising mucosal MCs.13
This could well be the case, as preliminary results from a 12-week
clinical trial indicate that ebastin, a selective H1R antagonist,
improves abdominal pain in IBS patients.14
H4 receptors (H4R) are currently under investigation for the
treatment of immune-mediated disorders as pruritus, asthma,
allergic rhinitis and rheumatoid arthritis.15 16 Present on immune
cells, among which MCs, on endocrine cells and on neurons,
H4Rs are expressed throughout the gastrointestinal tract.17 In
addition, pharmacological in vitro studies indicate that they are
functionally active in the human submucosal plexus.18 Moreover,
preliminary in vivo data demonstrate that in the GI tract, H4Rs
mediate indomethacin-induced gastric mucosal damage, modu-
late ischaemia/reperfusion-induced intestinal damage and
zymosan-induced peritonitis and participate in gut inflammation
in acute trinitrobenzenesulfonic acid (TNBS)-colitis.19–21 While
their role in inflammation has been extensively studied, H4R
involvement in visceral hypersensitivity has not been investigated
to date. In this study, we therefore aimed to explore the specific
contributions of H4Rs and H1Rs to visceral pain perception in a
rat model of postinflammatory visceral hypersensitivity.
METHODS
Animals
Male Sprague–Dawley rats (200–225 g, Charles River, France)
were allowed to acclimatise to housing conditions for 1 week
prior to experimentation. Animals had free access to water and
food and were kept at constant room temperature (22±2°C)
and humidity (60%) and on a 12:12 hour light/dark cycle. All
experiments were approved by the Committee for Medical
Ethics and the use of Experimental Animals at the University of
Antwerp (file number 2010-18).
Colitis
Distal colitis was induced by intrarectal instillation of 0.5 mL of
15 mg TNBS in 50% ethanol under pentobarbital anaesthesia
(60 mg/kg intraperitoneally). Control animals received a 0.5 mL
saline enema.
Colonoscopy
After pentobarbital sedation (45 mg/kg intraperitoneally), colon-
oscopy was performed using a baby gastroscope (Olympus
Europa GmbH, Germany). After lubrication and under direct
colonoscopic vision, the endoscope was gently introduced into
the distal 10 cm of the colon. During withdrawal, mucosal
damage was assessed using our previously published scoring
system (score 0–19).22 23
Postmortem evaluation of inflammatory markers
At the end of the experiment, macroscopic mucosal damage was
assessed using a standardised scoring system (score 0–10).22
A representative segment was fixed in 4% formaldehyde,
embedded in paraffin for H&E staining (5 mm) and scored
microscopically for the presence of an inflammatory infiltrate,
the number of layers infiltrated, mucosal architectural distortion
and oedema (score 0–10).22 The postinflammatory status was
additionally confirmed in paraffin-embedded sections by immu-
nohistochemical detection of CD3+ cells using anti-CD3
(Abcam; 1:300 dilution) as the primary antibody and a biotin-
conjugated antirabbit polyclonal secondary antibody. A colour
image analysis system was used to quantify the CD3-positive
area (ImageJ 1.47v, USA).
Myeloperoxidase (MPO) activity, which is directly related to
the number and activity of myeloid cell infiltration, was assayed
in colonic specimens as previously described in detail and
expressed as units per gram tissue (U/g tissue).23 24 Finally,
colonic levels of IL-2 and IL-17a were quantified by ELISA
(eBioscience, Austria) and expressed as pg/g tissue as these

Figure 1 Schematic of the experimental design. After the induction of trinitrobenzenesulfonic acid (TNBS) (colitis) or saline (control), animals were
monitored individually by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor
convalescence until full resolution of mucosal damage had occurred. Three days after resolution of colitis, further experiments were conducted. In
total, three experimental series were performed. Rats assigned to the first set did not receive drug or vehicle, while animals in the second and third
experimental groups were administered drug or vehicle 30 min prior to the start of the visceromotor response (VMR) distension protocol (set 2) or
30 min prior to sacrifice (set 3).

2 Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870

 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Neurogastroenterology

fully awake animals.23 28 Three days prior to VMR assessment,

Table 1 Confirmation of the postinflammatory status
two EMG electrodes (Advent Research Materials Ltd, UK) were
Control Postcolitis sutured into the external oblique abdominal muscle under deep
Marker (n=10) (n=10) pentobarbital anaesthesia (60 mg/kg) and exteriorised dorsally.
Colonoscopy (0–19)
On the day of VMR assessment, rats were mildly restrained using
On day 3 0.0±0.0 6.3±0.9*
a fabric glove. A lubricated, latex balloon (length 5 cm) was intro-
After exp 0.0±0.0 0.0±0.0
duced in the distal colon, up to 0.5 cm passed the anal verge and
Macroscopy (0–10) 0.0±0.0 0.0±0.0
connected to a barostat system (Distender Series II Barostat, G&J
Microscopy (0–10) 0.0±0.0 0.2±0.2
Electronics, Canada) for balloon distension (10–80 mm Hg, 20 s,
MPO (U/g tissue) 1.1±0.3 0.8±0.3
4 min interval). The electrodes were relayed to a data acquisition
CD3+ (% area)
system and the corresponding EMG signal was recorded, ampli-
Mucosa 1.7±0.3 1.3±0.3
fied (Neurolog, Digitimer Ltd, UK) and digitised (CED 1401,
Submucosa 0.5±0.2 0.7±0.2
Cambridge Electronic Design, UK) to a PC for off-line analysis
Muscularis 0.3±0.1 0.3±0.1
using Spike2 V.5.16 (Cambridge Electronic Design, UK). After
IL-2 (pg/g tissue) 118±31 90±7
correction for movement and breathing, the analogue EMG
IL-17a (pg/g tissue) 13±4 9±1
signal was rectified and integrated. To quantify the magnitude of
the VMR at each distension pressure, the area under the curve
Results are presented as mean±SEM. Unpaired Student t test.
*p<0.001, significantly different from control. (AUC) immediately before the distension (20 s) was subtracted
Exp, experiment; MPO, myeloperoxidase. from the AUC during the balloon distension (20 s).

cytokines have previously been shown to be upregulated during
acute TNBS-colitis.25–27
MCs and histamine release
A 1 cm colon segment was fixed in Carnoy’s solution and
embedded in paraffin. Transverse sections (5 mm) were stained
with toluidine blue ( pH 0.5) for identification of MCs.
The number of MCs was evaluated in 10 non-overlapping
fields (0.136 mm2/field) at ×400 magnification by an observer
blinded to the study groups and expressed per mm².
For in vitro quantification of the spontaneous histamine
release, a segment of distal colon (1 cm) was incubated in
Krebs–Ringer solution for 1 h at 37°C. Histamine levels in the
supernatant were evaluated by ELISA (Immunotech, France)
and expressed as nM/mg tissue.
Visceral sensitivity
The visceromotor response (VMR) to colorectal balloon disten-
sion was used as an objective measure of visceral sensitivity in
Colonic compliance
Colonic compliance was studied in the same animal to exclude
pharmacologically mediated changes in the viscoelastic proper-
ties of the colonic wall as a potential antinociceptive mechan-
ism. Rats were anesthetised ( pentobarbital 45 mg/kg) and
graded volumes (0–2.5 mL) were applied to the balloon inserted
in the colorectum while recording the corresponding intracolo-
nic pressure.23 29
Quantitative RT-PCR
The mRNA expression of H1R and H4R was quantified in the
colon and DRGs from controls and postcolitis rats. The DRGs
contain the primary afferent neurons conveying sensory infor-
mation from the colon to the spinal cord and were harvested
bilaterally at Th13-L2 (splanchnic afferents) and L6-S1 ( pelvic
afferents). Total RNA was extracted from colon using the
RNeasy Minikit (Qiagen, The Netherlands) and from DRGs
using the Absolutely RNA microprep kit (Stratagene, USA).
RNA was then converted to cDNA (Transcriptor First Strand
cDNA Synthese Kit; Roche, Belgium). A Taqman gene

Figure 2 (A) Toluidine blue staining demonstrating the presence of mast cells (*) in the colonic mucosa of a control and a postcolitis rat. (B) The
number of mast cells (MC) in the mucosa, submucosa and muscularis externa in controls (open bars) and after the resolution of
trinitrobenzenesulfonic acid (TNBS)-colitis (hatched bars). Two-way ANOVA followed by Student–Newman–Keuls posthoc test; n=6–7; ###p<0.001,
significant effect of the factor colitis; *p<0.05, significant effect of the factor layer, posthoc comparison; no significant interaction between layer and
colitis.

Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870 3

 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Neurogastroenterology

expression assay was performed for H1R (Rn00566691_s1;

Applied Biosystems, USA) and H4R (Rn00590929_m1; Applied
Biosystems, USA) on a ABIPrism 7300 sequent detector system
(Applied Biosystems) in a 25 mL reaction volume containing
2 mL cDNA, 12.5 mL TaqMan Universal PCR master mix
(Applied Biosystems), 1.25 mL Taqman assay probe and 9.25 mL
RNase-free H2O. The parameters for PCR amplification were
50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C
for 15 s and 60°C for 1 min.
Expression of H1R and H4R was normalised against the
housekeeping gene β-actin (Rn00667869_m1; Applied
Biosystems) for calculation of comparative cycle thresholds
(ΔCT=CT(H1R or H4R)—CT(β-actin)). Relative expression of
mRNA species was then determined as 2−ΔΔCT with ΔΔCT=ΔCT
( postcolitis)−ΔCT (control).30

Experimental design
A scheme of the experimental design is presented in figure 1.
Rats were randomised to receive a saline (control) or TNBS
instillation. The extent of colitis was colonoscopically verified
on day 3. From day 10 onwards, convalescence was monitored
individually by repeated colonoscopy that was performed every
4 days to determine for each individual animal whether endo-
scopic colonic healing had occurred. When mucosal healing was
present on colonoscopic examination, further experiments were
performed 3 days later. Three series of experiments were con-
ducted in three distinct sets of rats (figure 1).
In the first set of rats, colonic tissue and DRGs were harvested
3 days after endoscopic healing to quantify MC numbers, spon-
taneous histamine release and H4R and H1R mRNA expression.
Also, macroscopic and microscopic evaluation of inflammation
was performed to confirm the postinflammatory status in add-
ition to an MPO activity assay, CD3-staining and quantification
of IL-2 and IL-17a, which have previously been shown to be
increased during acute TNBS-colitis.25 31 N=10 in each group.
The second set of rats was injected intraperitoneally 3 days after
endoscopic healing (i) with JNJ7777120 (10–140 mg/kg; T1/2
≈2 h, bioavailability 22%), a selective H4R antagonist or its
vehicle (30% 2-hydroxypropyl-β-cyclodextrin); (ii) with levocetiri-
zine (0.01–1 mg/kg; T1/2 ≈8 h, bioavailability >90%), a selective
H1R antagonist or its vehicle (saline); or (iii) with a combination
of both antagonists ( JNJ7777120 10 mg/kg plus levocetirizine
0.1 mg/kg) or vehicle (30% 2-hydroxypropyl-β-cyclodextrin).
VMRs were assessed 30 min later. Colonic compliance was evalu-
ated immediately following the VMR distension protocol in rats
that had received the highest drug dose or its vehicle. At the end
of the experiments, rats were sacrificed to harvest colonic tissue
for macroscopic and microscopic analysis. N=4–9 in each group. Figure 3 (A) Spontaneous release of histamine from whole-mount
In a third set of rats, JNJ7777120 or levocetirizine, or their distal colon in controls and after the resolution of
respective vehicles, were administered 3 days after endoscopic trinitrobenzenesulfonic acid (TNBS)-colitis. Unpaired Student t test;
healing, and rats were sacrificed 30 min later. Spontaneous n=10; **p<0.01, significantly different from controls. (B) The effect of
colonic histamine release was assessed in vitro in addition to the JNJ7777120 (35 and 140 mg/kg) and its vehicle on spontaneous in
macroscopic and microscopic evaluation of the colon. N=8–10 vitro histamine release in the distal colon. Two-way ANOVA followed by
Student–Newman–Keuls posthoc test; n=9–10; significant interaction
in each group.
between colitis and drug; **p<0.01, significantly different from control
+vehicle. (C) The effect of levocetirizine (0.1 and 1 mg/kg) and its
Solutions and drugs vehicle on histamine release in vitro. Two-way ANOVA followed by SNK
JNJ7777120 was kindly gifted by Janssen Research & posthoc test; n=8–10; **p<0.01, significant effect of the factor colitis;
Development. Levocetirizine was purchased from UCB Pharma, no significant effect of the factor drug; no significant interaction.
Belgium. TNBS was bought from Fluka, Germany. Pentobarbital was
obtained from Ceva, Belgium. 2-Hydroxypropyl-β-cyclodextrin, Statistical analysis
hexadecyltrimethyl-ammonium bromide and o-dianisidine dihy- Data are presented as mean±SEM. Variables were analysed
drochloride were purchased from Sigma-Aldrich Inc, USA, and using unpaired Student t test and two-way ANOVA, followed by
hydrogen peroxide from Merck, Germany. Student–Newman–Keuls (SNK) posthoc test when appropriate.

4 Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870

 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com
Table 2 Postinflammatory status for animals receiving JNJ7777120 or vehicle
Control Postcolitis
Vehicle 70 mg/kg 140 mg/kg Vehicle
VMR (n=9) VMR (n=8) VMR (n=8) VMR (n=9)
Marker hist rel (n=10) hist rel (n=10) hist rel (n=10)
Colonoscopy
On day 3 0.0±0.0 0.0±0.0 0.0±0.0 7.1±0.8*
After exp 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Macroscopy 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Microscopy 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Data for each group are based on all animals included in the VMR and histamine release experiments. Results are presented as mean±SEM. Two-way ANOVA followed by posthoc SNK.
*p<0.001, significant effect of the factor colitis.
Exp, experiment; hist rel, histamine release; SNK, Student–Newman–Keuls; VMR, visceromotor response.
Analysis of VMR and compliance data was performed by the
generalised estimating equation model, followed by least signifi-
cant difference (LSD) posthoc test when appropriate. Statistical
analysis was executed using SPSS V.18.0 software. Statistical sig-
nificance was set at p<0.05.
RESULTS
MC numbers and histamine release are increased after the
resolution of TNBS-colitis
In the first set of experiments, a mild colitis characterised by
multiple serpiginous ulcers was present 3 days after
TNBS-instillation and resolved spontaneously after a mean
period of 11 days (range 10–22). Analysis of inflammation
markers (colonoscopy, macroscopy and microscopy, MPO
activity, CD3 expression and IL-2 and IL-17a levels) confirmed
the postinflammatory status at the time of the experiment
(table 1).
MCs were present in the colon of both control and postin-
flammatory rats with the following distribution: mucosa>sub-
mucosa>muscularis externa (figure 2A,B). The number of MCs
was significantly increased in the distal colon of postcolitis rats
compared with controls. In addition, the spontaneous release of
histamine was threefold higher in specimens obtained from post-
colitis rats compared with controls (figure 3A).
H4Rs contribute to postinflammatory visceral
hypersensitivity
After resolution of the TNBS-induced colitis (mean time to
mucosal healing 15 days; range 10–26 days), which was con-
firmed by colonoscopy and postmortem inflammatory markers
(table 2), VMRs were significantly increased in vehicle-treated
rats that had recovered from colitis compared with controls for
all distension pressures (10–80 mm Hg), indicating the presence
of postinflammatory visceral hypersensitivity (figure 4A). This
visceral hypersensitivity was dose-dependently reduced by
JNJ7777120 in postcolitis rats: 10 mg/kg had no significant
effect on VMRs, whereas 35 mg/kg effectively reversed
increased VMRs at the lower distension pressures (10 and
20 mm Hg) and 70 and 140 mg/kg normalised VMRs over the
full range of distension pressures (10–80 mm Hg) (figure 4A–
D). In contrast, in controls JNJ7777120 did not affect VMRs
significantly (figure 4E). Colonic compliance was similar in
control and postcolitis rats and was not modified by 140 mg/kg
of JNJ7777120 (figure 4F). In addition, JNJ7777120 treatment
did not affect the tissue’s colonoscopic, macroscopic and micro-
scopic appearance at any doses tested (table 2). In the distal
colon of postcolitis rats, H4R mRNA expression levels were
Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870
Neurogastroenterology
10 mg/kg 35 mg/kg 70 mg/kg 140 mg/kg
VMR (n=8) VMR (n=9) VMR (n=9) VMR (n=8)
hist rel (n=9) hist rel (n=10)
6.4±0.8* 7.0±0.8* 5.6±0.8* 6.9±0.8*
0.0±0.0 0.2±0.2 0.0±0.0 0.1±0.1
0.0±0.0 0.2±0.2 0.0±0.0 0.1±0.1
0.0±0.0 0.1±0.1 0.1±0.1 0.0±0.0
increased compared with controls (table 3); however, no H4R
mRNA could be demonstrated in the DRGs of either group.
The increased spontaneous release of histamine in the distal
colon of rats that had recovered from colitis remained equally
elevated after 35 mg/kg of JNJ7777120, whereas it was no
longer significantly different from controls after 140 mg/kg of
JNJ7777120 (figure 3B).
H1Rs contribute to postinflammatory visceral
hypersensitivity
Again, significant visceral hypersensitivity was present in rats that
had recovered from colitis (mean time to mucosal healing 13 days,
range 10–26 days) and were only vehicle-treated (figure 5A). This
postinflammatory hypersensitivity was not altered by the lowest
dose of levocetirizine (0.01 mg/kg), while 0.1 and 1 mg/kg nor-
malised heightened VMRs (figure 5A–C). The highest dose of
1 mg/kg did not affect visceral sensitivity in controls, nor did it
modify colonic compliance (figure 5D,E). Levocetirizine at the dif-
ferent doses tested did not influence postmortem inflammatory
parameters, which were in keeping with the postinflammatory
status (table 4).
The relative expression of H1R mRNA was comparable in the
colon and DRGs of control and postcolitis rats at Th13-L2 and
L6-S1 (table 3). Histamine release in the distal colon was signifi-
cantly increased in the postcolitis group and was not signifi-
cantly attenuated by levocetirizine, although histamine levels
tended to be decreased after the 1 mg/kg dose (figure 3C).
Combined H4/H1R antagonism potentiates the
antinociceptive effect
Significant visceral hypersensitivity, which was present after the
resolution of TNBS-colitis (mean time to mucosal healing
13 days, range 10–14 days), was significantly reduced by the
combined administration of 10 mg/kg of JNJ7777120 and
0.01 mg/kg of levocetirizine (figure 6A). In contrast, this treat-
ment did not modify VMRs in control, nor altered colonic com-
pliance (figure 6B,C). Postinflammatory markers were not
affected by the combined administration (table 5).
DISCUSSION
MCs, as key players of both the innate and adaptive immune
system, are powerful neuroimmune modulators implicated in
the regulation of various gastrointestinal functions in both
physiological and pathophysiological conditions. This study con-
firms their involvement in the pathogenesis of postinflammatory
visceral hypersensitivity and uncovers a substantial contribution
of H4Rs. Furthermore, our results confirm the potential role for
5
 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Neurogastroenterology

Figure 4 The effect of JNJ7777120 (10–140 mg/kg) on visceromotor responses (VMRs) in postcolitis (A–D) and control rats (E). To facilitate
comparison, VMRs for vehicle-treated controls are also shown in A–D (gray dashed line). Generalised estimating equations, least significant
difference posthoc test, n=8–9; #p<0.05, ##p<0.01, ###p<0.001, significantly different from control+vehicle. *p<0.05, **p<0.01, ***p<0.001,
significantly different from postcolitis+vehicle. (F) The effect of 140 mg/kg JNJ7777120 and its vehicle on colonic compliance in control and
postcolitis groups; no significant effect of the factor drug or colitis.

H1Rs in postinflammatory abdominal pain and reveal a func-
tional interplay between H4Rs and H1Rs.
Since the discovery of the H4R in 2000, promising results
have been published on the effect of H4R antagonists in animal
models for acute gastrointestinal inflammation induced by indo-
methacin, zymosan, TNBS and ischaemia/reperfusion.32 In add-
ition to its anti-inflammatory and immune modulating
properties, the selective H4R antagonist JNJ7777120 displayed
antiallodynic effects in two models of neuropathic pain—more
specifically a L5-L6 spinal nerve ligation and a chronic constric-
tion injury model—pointing towards an additional role for this
receptor subtype in nociception.21 We explored the analgesic
potential of H4R-targeted therapy in an in vivo model for post-
inflammatory visceral pain and showed that JNJ7777120 dose-
dependently reversed the hypersensitive VMRs to colorectal dis-
tension in postcolitis rats, without affecting visceral sensitivity in
controls. Importantly, we excluded that this antinociceptive
effect was mediated by changes in colonic compliance or by
altering the postinflammatory status. To our knowledge, this is
the first report demonstrating the involvement of the H4R
receptor in visceral hypersensitivity.
Low to high doses of JNJ7777120 were used in our set-up,
ranging from 10 up to 140 mg/kg, based on the compound’s
pharmacokinetic profile, previous reports in the literature and

6 Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870

 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Neurogastroenterology

the high affinity of histamine for the H4R compared with the
Table 3 mRNA expression of H4R and H1R in colon and dorsal
H1R.19 21 JNJ7777120 currently serves as the reference com-
root ganglias (Th13-L2 and L6-S1)
pound for H4R antagonists due to its high affinity for the H4R,
Control Postcolitis which is in the nanomolar range (Ki=4.2 nM for rat H4R).16 21
(n=10) (n=10)
In radio-ligand binding assays, JNJ7777120 displayed very high
H4R selectivity over the H1R (Ki >3000 nM), H2R (Ki >3000 nM)
Colon 1.15±0.20 2.00±0.27* and H3R (Ki >1000 nM) as well as over 50 other targets.21
Th13-L2 ND ND Moreover, in vivo studies administering up to 200 mg/kg
L6-S1 ND ND confirm the compound’s selectivity over the H1R.33 Therefore,
H1R we believe that the antinociceptive effects of the doses of
Colon 1.03±0.09 0.93±0.13 JNJ7777120 (10–140 mg/kg) we used are indeed mediated by
Th13-L2 1.89±0.78 0.96±0.35 inhibition of H4Rs.
L6-S1 1.13±0.19 1.23±0.26 In addition, our results demonstrate that levocetirizine, a
Results are presented as mean±SEM. selective H1R antagonist, also potently modulated postinflam-
Unpaired Student t test. matory visceral hypersensitivity in fully awake rats. These results
*p<0.05 significantly different compared with control.
ND, not determinable. are in agreement with a report by Stanisor et al34,

Figure 5 The effect of levocetirizine (0.01–1 mg/kg) on visceromotor responses (VMRs) in postcolitis (A–C) and control rats (D). To facilitate
comparison, VMRs for vehicle-treated controls are also shown in A–C (gray dashed line). generalised estimating equations, least significant
difference posthoc test, n=7–9; #<p<0.05, ##p<0.01, ###p<0.001, significantly different from control+vehicle. *p<0.05, **p<0.01, ***p<0.001,
significantly different from postcolitis+vehicle. (E) The effect of 1 mg/kg levocetirizine and its vehicle on colonic compliance in control and postcolitis
groups; no significant effect of the factor drug or colitis.

Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870 7

 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com
Neurogastroenterology
Table 4 Postinflammatory status for animals receiving levocetirizine or vehicle
Control
Vehicle 1 mg/kg
VMR (n=9) VMR (n=7)
Marker hist rel (n=10) hist rel (n=10)
Colonoscopy
On day 3 0.0±0.0 0.0±0.0
After exp 0.0±0.0 0.0±0.0
Macroscopy 0.0±0.0 0.0±0.0
Microscopy 0.0±0.0 0.0±0.0
Data for each group are based on all animals included in the VMR and histamine release experiments. Results are presented as mean±SEM. Two-way ANOVA followed by posthoc SNK.
*p<0.001, significant effect of the factor colitis.
Exp, experiment; hist rel, histamine release; SNK, Student–Newman–Keuls; VMR, visceromotor response.
demonstrating reversal of stress-induced visceral hypersensitivity
by fexofenadine, another H1R antagonist, in a rat model of
maternal separation. Our data also coincide with a preliminary
report that ebastin, yet another selective H1R antagonist,
improves abdominal pain in IBS patients.14
Visceral hypersensitivity can result from peripheral sensitisa-
tion of afferent nerves, spinal facilitation through sensitisation
of dorsal horn neurons, dysfunction of descending excitatory or
inhibitory pathways or from altered central pain processing.35
As H4Rs and H1Rs are expressed on multiple levels of the pain
signalling pathways, both peripheral and/or central mechanisms
could be involved in the antinociceptive effects of our H4R and
H1R antagonists.
As JNJ7777120 can readily cross the blood–brain barrier, a cen-
trally mediated mechanism is possible.21 Indeed, immunohisto-
chemistry previously revealed the presence of H4Rs in murine
spinal cord, cortex, amygdala, hippocampus and thalamus, of
which the latter three have been implicated in altered central pro-
cessing of pain signals in IBS patients.36–39 To date, it remains
uncertain whether and to what extent H4Rs are involved in con-
veying sensory information from the gut to the central nervous
system. Previously, histamine-induced jejunal afferent firing in
vitro was not affected by a combined H3R/H4R antagonist and in
a recent study by Kajihara et al40 JNJ7777120 did not reduce
histamine-induced excitation of DRGs.41 However, RT-PCR ana-
lysis did reveal expression of the H4R subtype in mice DRGs.39 40
Unfortunately none of these studies on DRGs indicate the spinal
level at which they were harvested.39 40 On the other hand, it
remains to be established whether or not these data, gathered in
mice models, also pertain to rat colonic afferents. Either way, in
our model we were not able to demonstrate H4R mRNA expres-
sion in the DRGs containing the pelvic and splanchnic afferent
neuronal cell bodies, suggesting the mode of action for
JNJ7777120 might not take place at the DRG level. To further
elucidate a peripheral mechanism, we evaluated the effects of
JNJ7777120 on spontaneous histamine release from the colon.
Histamine levels were threefold increased after the resolution of
colitis, remained high after an intermediate dose of JNJ7777120
(35 mg/kg) but were no longer different from controls after the
highest dose (140 mg/kg). These results suggest a peripheral mech-
anism for JNJ7777120 that interconnects with the postinflamma-
tory state as the compound impedes excessive histamine release
only in the colon of postcolitis rats, probably resulting in a reduc-
tion of colorectal distension-induced nociception.
For the H1R antagonist, a central mechanism of action seems
unlikely as levocetirizine shows limited blood–brain barrier
penetration.42 In addition, levocetirizine is devoid of central
side effects at the clinical daily dose for treatment of allergic
8 Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870
Post-colitis
Vehicle 0.01 mg/kg 0.1 mg/kg 1 mg/kg
VMR (n=9) VMR (n=7) VMR (n=7) VMR (n=7)
hist rel (n=10) hist rel (n=8) hist rel (n=9)
7.3±0.5* 7.6±0.6* 6.7±0.6* 7.4±0.6*
0.1±0.1 0.0±0.0 0.0±0.0 0.2±0.2
0.1±0.1 0.0±0.0 0.1±0.1 0.1±0.1
0.0±0.0 0.0±0.0 0.0±0.0 0.1±0.1
rhinitis (5 mg), which is roughly equivalent to the 0.1 mg/kg
dose that effectively reduced VMRs in our study.42 Besides it is
generally accepted that peripheral histamine stimulates nocicep-
tive nerve fibres via activation of H1Rs.43 In particular for vis-
ceral perception, the application of histamine or IBS
supernatant containing excessive amounts of MC mediators
enhanced mesenteric sensory nerve firing in murine-afferent
recordings and stimulated the [Ca2+]i mobilisation in isolated
DRGs. These excitatory effects were inhibited by application of
a H1R antagonist in all these studies.8 41 44 The heightened
VMRs in our study that were blocked by levocetirizine seem in
line with this previous evidence.
We found arguments for a differential mechanism of action
for H1R and H4R antagonists. In contrast to H4R, the colonic
H1R mRNA expression level was similar in postinflammatory
and control conditions; in addition, H1R but not H4R mRNA
was present in the relevant DRGs. These findings seem to
concur with the possibility of histamine acting on H1Rs on
afferent nerve fibres at the level of the colon and/or the DRG,
whereas the activation of H4R seems to occur in the colonic
wall, where H4Rs were previously described on intestinal MCs,
enteroendocrine cells and immunocytes.17 43
This differential mechanism of action most likely provides the
basis for the functional interplay between both receptor sub-
types as evidenced by the potentiation of the antinociceptive
effect by simultaneous administration of both antagonists. This
finding also implies that combination therapy might represent a
potent and possibly safe(r) treatment strategy as drug dose
reduction most likely results in fewer side effects.
Our findings undoubtedly corroborate the involvement of his-
tamine in visceral hypersensitivity. Interestingly, similar results
have been reported for other MC mediators such as tryptase
acting on protease-activated receptors PAR2 and PAR4 and for
serotonin, released from enterochromaffin cells located in the
gut mucosa.9 45–48 This leads to the hypothesis that histamine,
tryptase and serotonin might act as sensitising agents modulat-
ing a common target. The transient receptor potential vanilloid
(TRPV) family is a likely candidate for such a common target.49
In that regard, histamine, tryptase and serotonin have been
shown to sensitise TRPV1 and TRPV4 by inducing receptor
phosphorylation, translocation and potentiation.49–51
Preliminary data mainly implicate the H1R subtype in this sensi-
tising effect of histamine on TRPV1 and TRPV4, with very little
data available on its H4R counterpart, warranting further
study.40 49
In summary, considering the emerging role for MCs and their
main mediator histamine in the pathogenesis of IBS, we investi-
gated the contribution of H4Rs and H1Rs to postinflammatory
 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Neurogastroenterology

Table 5 Postinflammatory status for animals receiving combined

treatment with JNJ7777120 (10 mg/kg) and levocetirizine (0.01 mg/
kg) or vehicle
Control Postcolitis
Vehicle H4/H1 Vehicle H4/H1
Marker VMR (n=4) VMR (n=4) VMR (n=7) VMR (n=7)

Colonoscopy
On day 3 0.0±0.0 0.0±0.0 4.9±0.5* 6.0±0.5*
After exp 0.0±0.0 0.0±0.0 0.2±0.2 0.0±0.0
Macroscopy 0.0±0.0 0.0±0.0 0.0±0.0 0.1±0.1
Microscopy 0.0±0.0 0.0±0.0 0.0±0.0 0.2±0.1
Data for each group are based on all animals included in the VMR experiments.
Results are presented as mean±SEM. Two-way ANOVA followed by posthoc SNK.
*p<0.001, significant effect of the factor colitis.
Exp, experiment; hist rel, histamine release; SNK, Student–Newman–Keuls; VMR,
visceromotor response.

for postinflammatory visceral hypersensitivity, with a functional

interplay between H4Rs and H1Rs. mRNA expression and, to a
lesser extent, histamine release data point towards a different
mode of action for both drugs. To conclude, our results reveal
that both H4Rs and H1Rs play a substantial role in postinflam-
matory visceral abdominal pain and bare great potential for spe-
cific histamine receptor subtype-targeted therapy in the
treatment of postinflammatory visceral hypersensitivity. As H4R
antagonists have already entered phase II clinical trials for
asthma, clinical studies investigating their effect on IBS symp-
toms could be initiated in the short term. In addition, our
results strongly support the rationale for continuation of clinical
trials with H1R antagonists in IBS.

Acknowledgements We would like to thank Janssen Research & Development for

supplying JNJ7777120 and Dr R Thurmond for the critical review of the manuscript.
A special thanks to our lab technicians, P Aerts, A Jürgens, M Vinckx, A Van Daele
and R Van Den Bossche, for their technical assistance.
Contributors All authors contributed to the conception and design of the study,
interpreted the data, revised the manuscript critically for important intellectual
content and approved the final version. In addition, AD collected the data, and AD,
JGDM and BYDW drafted the manuscript.
Funding AD is an aspirant of the Fund for Scientific Research (FWO), Flanders. This
work was supported financially by the FWO (G.0341.13 and G.0249.09N).
Competing interests None.
Patient consent Obtained.
Ethics approval Committee for Medical Ethics and the use of Experimental
Animals at the University of Antwerp.
Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES
Figure 6 The effect of combined administration of JNJ7777120
(10 mg/kg) and levocetirizine (0.01 mg/kg) on visceromotor responses
(VMRs) in postcolitis (A) and control rats (B). To facilitate comparison,
VMRs for vehicle-treated controls are also shown in (A) (gray dashed
line). Generalised estimating equations, least significant difference
posthoc test, n=4–7; #p<0.05, ##p<0.01, ###p<0.001, significantly
different from control+vehicle. *p<0.05, **p<0.01, significantly
different from post-colitis+vehicle. (C) The effect of combined
administration of 10 mg/kg JNJ7777120 and 0.01 mg/kg levocetirizine
on colonic compliance in control and postcolitis groups; no significant
effect of the factor drug or colitis.
visceral hypersensitivity. Both the selective H4R antagonist
JNJ7777120 and the selective H1R antagonist levocetirizine nor-
malised increased VMRs to colorectal distension in a rat model
1 Barbara G, Cremon C, De Giorgio R, et al. Mechanisms underlying visceral
hypersensitivity in irritable bowel syndrome. Curr Gastroenterol Rep
2011;13:308–15.
2 Truong TT, Naliboff BD, Chang L. Novel techniques to study visceral hypersensitivity
in irritable bowel syndrome. Curr Gastroenterol Rep 2008;10:369–78.
3 Lembo T, Naliboff B, Munakata J, et al. Symptoms and visceral perception in
patients with pain-predominant irritable bowel syndrome. Am J Gastroenterol
1999;94:1320–6.
4 Bulmer DC, Grundy D. Achieving translation in models of visceral pain. Curr Opin
Pharmacol 2011;11:575–81.
5 Van Nassauw L, Adriaensen D, Timmermans JP. The bidirectional communication
between neurons and mast cells within the gastrointestinal tract. Auton Neurosci
2007;133:91–103.
6 De Winter BY, van den Wijngaard RM, de Jonge WJ. Intestinal mast cells in gut
inflammation and motility disturbances. Biochim Biophys Acta 2012;1822:66–73.
7 Barbara G, Stanghellini V, De Giorgio R, et al. Activated mast cells in proximity to
colonic nerves correlate with abdominal pain in irritable bowel syndrome.
Gastroenterology 2004;126:693–702.

Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870 9

 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com
Neurogastroenterology
8 Barbara G, Wang B, Stanghellini V, et al. Mast cell-dependent excitation of
visceral-nociceptive sensory neurons in irritable bowel syndrome. Gastroenterology
2007;132:26–37.
9 Cenac N, Andrews CN, Holzhausen M, et al. Role for protease activity in visceral
pain in irritable bowel syndrome. J Clin Invest 2007;117:636–47.
10 Buhner S, Li Q, Vignali S, et al. Activation of human enteric neurons by
supernatants of colonic biopsy specimens from patients with irritable bowel
syndrome. Gastroenterology 2009;137:1425–34.
11 Buhner S, Li Q, Braak B, et al. Excitation of enteric neurons by supernatans of
colonic biopsies from irritable bowel syndrome patients (IBS) is linked to visceral
hypersensitivity. Gastroenterology 2011;140:S521.
12 Klooker TK, Braak B, Koopman KE, et al. The mast cell stabiliser ketotifen decreases
visceral hypersensitivity and improves intestinal symptoms in patients with irritable
bowel syndrome. Gut 2010;59:1213–21.
13 Wouters MM, Boeckxstaens GE, Klooker TK, et al. Reply. Gastroenterology
2011;140:2136.
14 van Wanrooij S, Wouters MM, Van Oudenhove L, et al. Effect of the H1-receptor
antagonist ebastin on visceral perception and clinical symptoms in IBS.
Gastroenterology 2013;144:S-160.
15 Zampeli E, Tiligada E. The role of histamine H4 receptor in immune and
inflammatory disorders. Br J Pharmacol 2009;157:24–33.
16 Smits RA, Leurs R, de Esch IJ. Major advances in the development of histamine H4
receptor ligands. Drug Discov Today 2009;14:745–53.
17 Sander LE, Lorentz A, Sellge G, et al. Selective expression of histamine receptors
H1R, H2R, and H4R, but not H3R, in the human intestinal tract. Gut
2006;55:498–504.
18 Breunig E, Michel K, Zeller F, et al. Histamine excites neurones in the human
submucous plexus through activation of H1, H2, H3 and H4 receptors. J Physiol
2007;583:731–42.
19 Adami M, Pozzoli C, Menozzi A, et al. Effects of histamine H4 receptor ligands in a
mouse model of gastric ulceration. Pharmacology 2012;89:287–94.
20 Varga C, Horvath K, Berko A, et al. Inhibitory effects of histamine H4 receptor
antagonists on experimental colitis in the rat. Eur J Pharmacol 2005;522:130–8.
21 Hsieh GC, Chandran P, Salyers AK, et al. H4 receptor antagonism exhibits
anti-nociceptive effects in inflammatory and neuropathic pain models in rats.
Pharmacol Biochem Behav 2010;95:41–50.
22 Vermeulen W, De Man JG, Nullens S, et al. The use of endoscopy to follow the
inflammatory time course of TNBS-colitis in rats. Acta Gastroenterol Belg
2011;74:304–11.
23 Vermeulen W, De Man JG, De Schepper HU, et al. Role of TRPV1 and TRPA1 in
visceral hypersensitivity to colorectal distension during experimental colitis in rats.
Eur J Pharmacol 2013;698:404–12.
24 Heylen M, Deleye S, De Man JG, et al. Colonoscopy and microPET/CT are valid
techniques to monitor inflammation in the adoptive transfer colitis model in mice.
Inflamm Bowel Dis 2013;19:967–76.
25 Moreels TG, Nieuwendijk RJ, De Man JG, et al. Concurrent infection with
Schistosoma mansoni attenuates inflammation induced changes in colonic
morphology, cytokine levels, and smooth muscle contractility of trinitrobenzene
sulphonic acid induced colitis in rats. Gut 2004;53:99–107.
26 Zhu MY, Lu YM, Ou YX, et al. Dynamic progress of 2,4,6-trinitrobenzene sulfonic
acid induced chronic colitis and fibrosis in rat model. J Dig Dis 2012;13:421–9.
27 Daniel C, Sartory NA, Zahn N, et al. Immune modulatory treatment of
trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a
T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile. J Pharmacol Exp Ther
2008;324:23–33.
28 Ness TJ, Gebhart GF. Colorectal distension as a noxious visceral stimulus:
physiologic and pharmacologic characterization of pseudaffective reflexes in the rat.
Brain Res 1988;450:153–69.
29 De Schepper HU, De Winter BY, Van Nassauw L, et al. TRPV1 receptors on
unmyelinated C-fibres mediate colitis-induced sensitization of pelvic afferent nerve
fibres in rats. J Physiol 2008;586:5247–58.
10
30 Abad C, Juarranz Y, Martinez C, et al. cDNA array analysis of cytokines,
chemokines, and receptors involved in the development of TNBS-induced colitis:
homeostatic role of VIP. Inflamm Bowel Dis 2005;11:674–84.
31 Alex P, Zachos NC, Nguyen T, et al. Distinct cytokine patterns identified from
multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm Bowel Dis
2009;15:341–52.
32 Coruzzi G, Adami M, Pozzoli C. Role of histamine H4 receptors in the
gastrointestinal tract. Front Biosci (Schol Ed) 2012;4:226–39.
33 Thurmond RL, Desai PJ, Dunford PJ, et al. A potent and selective histamine H4
receptor antagonist with anti-inflammatory properties. J Pharmacol Exp Ther
2004;309:404–13.
34 Stanisor OI, van Diest SA, Welting O, et al. The peripheral histamine 1-receptor
antagonist fexofenadine effectively reverses stress-induced visceral hypersensitivity in
a rat model of maternal separation. Gastroenterology 2010;138:S1.
35 Anand P, Aziz Q, Willert R, et al. Peripheral and central mechanisms of visceral
sensitization in man. Neurogastroenterol Motil 2007;19:29–46.
36 Connelly WM, Shenton FC, Lethbridge N, et al. The histamine H4 receptor is
functionally expressed on neurons in the mammalian CNS. Br J Pharmacol
2009;157:55–63.
37 Katabe M, Chazot PL. The histamine H4 receptor is strongly expressed on a subset
of Aδ sensory fiberts at the level of rat skin, DRG and dorsal horn of the spinal
cord. Inflamm Res 2012;61:S60.
38 Mayer EA, Aziz Q, Coen S, et al. Brain imaging approaches to the study of
functional GI disorders: a Rome working team report. Neurogastroenterol Motil
2009;21:579–96.
39 Strakhova MI, Nikkel AL, Manelli AM, et al. Localization of histamine H4
receptors in the central nervous system of human and rat. Brain Res
2009;1250:41–8.
40 Kajihara Y, Murakami M, Imagawa T, et al. Histamine potentiates acid-induced
responses mediating transient receptor potential V1 in mouse primary sensory
neurons. Neuroscience 2010;166:292–304.
41 Kreis ME, Haupt W, Kirkup AJ, et al. Histamine sensitivity of mesenteric afferent
nerves in the rat jejunum. Am J Physiol 1998;275:G675–80.
42 Verster JC, de Weert AM, Bijtjes SI, et al. Driving ability after acute and sub-chronic
administration of levocetirizine and diphenhydramine: a randomized, double-blind,
placebo-controlled trial. Psychopharmacology 2003;169:84–90.
43 Brown RE, Stevens DR, Haas HL. The physiology of brain histamine. Prog Neurobiol
2001;63:637–72.
44 Brunsden AM, Grundy D. Sensitization of visceral afferents to bradykinin in rat
jejunum in vitro. J Physiol 1999;521:517–27.
45 Greenwood-Van Meerveld B, Venkova K, Hicks G, et al. Activation of peripheral
5-HT receptors attenuates colonic sensitivity to intraluminal distension.
Neurogastroenterol Motil 2006;18:76–86.
46 O’Mahony SM, Bulmer DC, Coelho AM, et al. 5-HT(2B) receptors modulate visceral
hypersensitivity in a stress-sensitive animal model of brain-gut axis dysfunction.
Neurogastroenterol Motil 2010;22:573–8, e124.
47 Ohashi-Doi K, Himaki D, Nagao K, et al. A selective, high affinity 5-HT 2B receptor
antagonist inhibits visceral hypersensitivity in rats. Neurogastroenterol Motil
2010;22:e69–76.
48 Auge C, Balz-Hara D, Steinhoff M, et al. Protease-activated receptor-4 (PAR 4): a
role as inhibitor of visceral pain and hypersensitivity. Neurogastroenterol Motil
2009;21:1189–e107.
49 Cenac N, Altier C, Motta JP, et al. Potentiation of TRPV4 signalling by histamine
and serotonin: an important mechanism for visceral hypersensitivity. Gut
2010;59:481–8.
50 Grant AD, Cottrell GS, Amadesi S, et al. Protease-activated receptor 2 sensitizes the
transient receptor potential vanilloid 4 ion channel to cause mechanical
hyperalgesia in mice. J Physiol 2007;578:715–33.
51 Amadesi S, Nie J, Vergnolle N, et al. Protease-activated receptor 2 sensitizes the
capsaicin receptor transient receptor potential vanilloid receptor 1 to induce
hyperalgesia. J Neurosci 2004;24:4300–12.
Deiteren A, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2013-305870
 Downloaded from gut.bmj.com on February 22, 2014 - Published by group.bmj.com

Histamine H4 and H1 receptors contribute to

postinflammatory visceral hypersensitivity
Annemie Deiteren, Joris G De Man, Nathalie E Ruyssers, et al.

Gut published online February 21, 2014

doi: 10.1136/gutjnl-2013-305870

Updated information and services can be found at:

http://gut.bmj.com/content/early/2014/02/21/gutjnl-2013-305870.full.html

These include:
References This article cites 51 articles, 12 of which can be accessed free at:
http://gut.bmj.com/content/early/2014/02/21/gutjnl-2013-305870.full.html#ref-list-1

P<P Published online February 21, 2014 in advance of the print journal.

Email alerting Receive free email alerts when new articles cite this article. Sign up in
service the box at the top right corner of the online article.

Topic Articles on similar topics can be found in the following collections

Collections
Gastrointestinal hormones (836 articles)
Colon cancer (1301 articles)
Endoscopy (798 articles)

Notes

Advance online articles have been peer reviewed, accepted for publication, edited and
typeset, but have not not yet appeared in the paper journal. Advance online articles are
citable and establish publication priority; they are indexed by PubMed from initial
publication. Citations to Advance online articles must include the digital object identifier
(DOIs) and date of initial publication.

To request permissions go to:

http://group.bmj.com/group/rights-licensing/permissions

To order reprints go to:

http://journals.bmj.com/cgi/reprintform

To subscribe to BMJ go to:

http://group.bmj.com/subscribe/