Peripheral α-helical CRF (9-41) does not reverse stress-induced mast cell dependent visceral...

Peripheral a-helical CRF (9-41) does not reverse stress-

induced mast cell dependent visceral hypersensitivity

in maternally separated rats

R. M. VAN DEN WIJNGAARD,*1O. I. STANISOR,*1

S. A. VAN DIEST,* O. WELTING,* M. M. WOUTERS,� W. J. DE JONGE*& G. E. BOECKXSTAENS*,�

*Tytgat Institute for Liver and Intestinal research, Academic Medical Center, Amsterdam, The Netherlands

�Division of Gastroenterology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium

Abstract

Background Acute stress-induced hypersensitivity to

colorectal distention was shown to depend on corti-

cotropin releasing factor (CRF)-induced mast cell

degranulation. At present it remains unclear whether

CRF also induces chronic poststress activation of

these cells. Accordingly, the objective of this study

was to compare pre- and poststress CRF-receptor

antagonist treatment protocols for their ability to,

respectively, prevent and reverse mast cell dependent

visceral hypersensitivity in a rat model of neonatal

maternal separation. Methods The visceromotor

response to colonic distention was assessed in adult

maternally separated and non-handled rats before and

at different time points after 1 h of water avoidance

(WA). Rats were treated with the mast cell stabilizer

doxantrazole and the CRF receptor-antagonist a-heli-

cal-CRF (9-41). Western blotting was used to assess

mucosal protein levels of the mast cell protease

RMCP-2 and the tight junction protein occludin. Key

Results In maternally separated, but not in non-

handled rats, WA induced chronic hypersensitivity

(up to 30 days) to colorectal distention. Visceral

hypersensitivity was prevented, but could not be

reversed by administration of a-helical-CRF (9-41). In

contrast, however, the mast cell stabilizer doxantraz-

ole reversed visceral hypersensitivity. Compared with

vehicle-treated rats, pre-WA a-helical-CRF (9-41)

treated animals displayed higher mucosal RMCP-2

and occludin levels. Conclusions & Inferences Water

avoidance-stress leads to persistent mast cell depen-

dent visceral hypersensitivity in maternally separated

rats, which can be prevented, but not reversed by

blockade of peripheral CRF-receptors. We conclude

that persistent poststress mast cell activation and

subsequent visceral hypersensitivity are not targeted

by CRF-receptor antagonists.

Keywords corticotropin-releasing-factor, irritable

bowel syndrome, mast cells, rat, stress, visceral

hypersensitivity.

Abbreviations: AUC, area-under-curve; CRF, corticotro-

pin-releasing-factor; IBS, irritable bowel syndrome;

WA, water avoidance.

INTRODUCTION

The irritable bowel syndrome (IBS) is a functional

bowel disorder characterized by abdominal pain or

discomfort associated with defecation or change in

bowel habit.1 Stress plays an important role in the

onset and modulation of IBS. It induces increased

perception of gastro-intestinal stimuli, so called vis-

ceral hypersensitivity, which is thought to be an

important pathophysiological mechanism in this dis-

order.2,3 In animal models, stress not only leads to

visceral hypersensitivity, but also induces intestinal

permeability changes.4,5 Ex vivo studies in patients

also showed intestinal barrier dysfunction and related

changes in expression of tight junction proteins (zonula

occludens (ZO)-1 and occludin).4,6–9 Animal experi-

ments indicated that stress-induced barrier and sensi-

tivity changes may be caused by activation of

intestinal mucosal mast cells.4,5,10 Investigations per-

formed with supernatants of submerged intestinal

Address for Correspondence

Rene M van den Wijngaard, Tytgat Institute for Liver andIntestinal Research, Academic Medical Center, Room S2-180,Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands.Tel: +31 020 566 8163; fax: +31 020 566 9190;e-mail: [email protected] authors contributed equally to this work.Received: 1 September 2011Accepted for publication: 31 October 2011

Neurogastroenterol Motil (2012) 24, 274–e111 doi: 10.1111/j.1365-2982.2011.01840.x

� 2011 Blackwell Publishing Ltd274

Neurogastroenterology & Motility

biopsies from IBS patients and normal controls con-

firmed that these cells may indeed be relevant.11,12

Recently, a possible in vivo role for mast cells was

corroborated in a double-blind placebo-controlled

patient trial conducted in our own laboratory. The mast

cell stabilizer and H1-receptor antagonist ketotifen

reduced threshold of discomfort and IBS-symptoms

and improved health related quality of life.13 Next to

the use of mast cell stabilizers, targeting of degranu-

lation triggers may also be a treatment option for IBS.

In preclinical investigations it was shown that stress-

induced IBS-like phenotypical changes (visceral hyper-

sensitivity and barrier dysfunction) are mediated by

corticotropin releasing factor (CRF)4,5,14 and, conse-

quently, the possible role of CRF-mediated mast cell

degranulation was investigated. Hypersensitivity to

distention, induced by partial restraint stress, was

mimicked by intracerebroventricular CRF-administra-

tion and prevented when rats were pretreated with

central CRF-receptor antagonist or peripheral mast cell

stabilizer.15 Importantly, chronic subcutaneous CRF

administration in normal (+/+) and mast cell deficient

(Ws/Ws)-rats indicated that, next to central, also periph-

eral CRF may be relevant: CRF treatment resulted in

barrier dysfunction in +/+ but not Ws/Ws rats.16 These

results were confirmed when CRF was added ex vivo in

Ussing-chamber experiments with mast cell-sufficient

and -deficient colonic rat tissue.17 Finally, Ussing-

experiments performed by Wallon et al. indicated that

human colonic mast cells are also susceptible to CRF-

mediated mast cell degranulation.18 Although this

cumulative evidence suggests that CRF-receptors are

an attractive therapeutical target in IBS, it is important

to note that most studies were aimed at prevention of

stress-induced phenotypical changes4,5,14 whereas

reversal may be more relevant to patients. At present

it remains unclear whether post-stress receptor-antag-

onist treatment is able to reverse mast cell dependent

visceral hypersensitivity or, alternatively, other triggers

induce chronic poststress mast cell activation. Accord-

ingly, the objective of this study was to compare pre-

and poststress CRF-receptor antagonist treatment pro-

tocols for their ability to, respectively, prevent and

reverse mast cell dependent visceral hypersensitivity.

These investigations were carried out in the maternal

separation model for rats in which stress-induced mast

cell mediated IBS-like phenotypical changes are well

described characteristics.10,19

MATERIAL AND METHODS

All protocols were approved by the Ethical Animal ResearchCommittee of the University of Amsterdam.

Animals

Long-Evans rats (Harlan, Horst, The Netherlands) were kept instandard macralon cages with a layer of wood shavings and housedat the animal facility of the AMC (Amsterdam, The Netherlands)under conditions of controlled light (06:00–18:00), temperature(20–22 �C), and humidity (45%). Water and food (SDS; TechnilabBMI, Someren, The Netherlands) were available ad libitum. Non-handled and maternally separated animals were bred in our ownanimal facilities.

Maternal separation protocol

Primiparous pregnant rats reared non-handled male pups; secondtime pregnant dams reared male pups that were subjected to thematernal separation protocol. During separation, dams wereplaced in another cage in a separate room for 3 h day)1 frompostnatal day 2 to 14. Meanwhile, litter was not removed from thenest, but left undisturbed except for placing of infrared light (27–30 �C). Non-handled pups were nursed normally. Maternallyseparated and non-handled pups were weaned on postnatal day22 and housed in pairs of two.

Measurement of the visceromotor responseto colorectal distention and data analysis

Distention of the colon induces contractions of abdominalmusculature: the so called visceromotor response. In rodents, itsquantitation by means of electromyography (EMG) is often usedas a surrogate measure for visceral sensitivity. We previouslyvalidated a radio-telemetry technique in freely moving rats torecord these signals.20 This technique does not require restraintduring EMG measurements, herewith limiting unwanted stressresponses that may obscure pre- and post-WA data sets. Furtherdetails on techniques and data analysis system have beenpublished extensively.19–21 Similar to other publications,20,22 finalresults are evaluated from normalized data sets, which werecalculated from the absolute data by setting the 2 mL value of thefirst (pre-WA) distentions (1.0, 1.5, and 2.0 mL) of each rat at100%. These relative response data were used to evaluate possiblechanges on a per volume basis. In addition, area under the curve(AUC) of relative responses was calculated for individual rats andalso used to show possible changes in visceromotor responsewithin treatment groups.

Colonic distention protocol and wateravoidance

Colonic distentions were performed with a latex balloon (Ultra-cover 8F, International Medical Products, Zutphen, The Nether-lands) and carried out as described before.20 Distentions wereperformed at the minimum age of 3 months by inflation of gradedvolumes of water (1.0, 1.5, and 2.0 mL) and started 20 min afterthe catheter was inserted under brief isoflurane anesthesia.Length and diameter of the balloon during maximum volumedistention were 18 mm and 15 mm, respectively. After each 20-sdistention episode water was quickly removed and an 80-s restingperiod was exercised. Possible pharmacological effects on compli-ance were assessed by determining the pressure–volume relation-ship in a subset of separated rats (carried out as described in ourprevious publications19,21). For acute stress at adult age, we used1 h of water avoidance (WA), during which rats are positioned on apedestal surrounded by water. It suffices to induce enhanced

Volume 24, Number 3, March 2012 CRF in poststress visceral hypersensitivity

� 2011 Blackwell Publishing Ltd 275

sensitivity to colonic distention in maternally separated ratswhich is not observed in the absence of water.20

Experimental design of pharmacologicalintervention studies

All animal experiments were performed by the same investigator(OW) who was blinded to administration of drug or vehicle alone(disclosed after evaluation of all tracings). Fig. 1A–C provides aschematic representation of all pharmacological interventionprotocols described below.

Reversal of chronic visceral hypersensitivity by mast cell stabi-

lizer doxantrazole: treatment between post-WA day 30 and

31 After measuring their baseline sensitivity to distention, fourgroups of rats (two non-handled and two maternally separated,n = 10/group) were subjected to WA and subsequent distentionprotocols at post-WA day 1, 5, 18, and 30. Directly after disten-tions at day 30 (09.00 AM) the different groups were treated(intraperitoneally) with either the mast cell stabilizer doxantraz-ole (10 mg kg)1, gift of Agnes Francois, Institut Gustave Roussy,Villejuif, France) which was dissolved in 0.5% NaHCO3/0.9%saline pH 7.5, or vehicle alone. i.p. treatments were repeated at06.00 PM (day 30) and 09.00 AM of day 31. Thirty minutes later ratswere subjected to the last distention protocol and sacrificeddirectly after.

Prevention of acute (and chronic) visceral hypersensitivity with

CRF-receptor antagonist a-helical-CRF(9-41): pre-WA treat-

ment The non-selective CRF-receptor antagonist a-helical-CRF(9-41) does not cross the blood-brain barrier (BBB). We adminis-tered 250 lg kg)123 (Tocris, Bristol, UK) or vehicle (saline) alone(i.p.) to maternally separated rats (n = 9/group) 30 min before thepre-WA distention protocol at 08.30 AM. Post-WA distentionswere carried out at T + 6 h and T + 23 days. Colonic tissue wascollected after sacrifice at T + 30 days.

Reversal of chronic visceral hypersensitivity with a-helical-CRF

(9-41): treatment between post-WA day 15 and 16 Baseline sen-sitivity to distention was measured in two groups of maternallyseparated rats (n = 9/group) which were then subjected to WA.Post-WA distentions were performed at T + 15, T + 16, andT + 23 days (at 09.00 AM). The CRF-receptor-antagonist (or vehiclealone) was administered three times (250 lg kg)1 per i.p.

injection) in between distentions at T + 15 days (09.30 AM & 18.00PM) and T + 16 days (08.30 AM). Colonic tissue was collected aftersacrifice at T + 30 days.

Western blotting

Stripped mucosa was obtained from distal colon, homogenized inlysis buffer (Cell Signaling, Danvers, MA, USA) and assessed bySDS-polyacrylamide gel electrophoresis and Western blotting.Blots were cut at appropriate kD and evaluated for expression ofthe rat chymase analog RMCP-2 (polyclonal anti-RMCP-2, More-dun Scientific, Penicuik, Scotland), the tight junction proteinoccludin (rabbit-anti-occludin, Zymed, San Francisco, CA, USA)and GAPDH (mouse-anti-GAPDH, Millipore, Amsterdam, TheNetherlands). Peroxidase-labeled secondary antibody was visual-ized with Lumi-light plus (Roche Diagnostics, Almere, TheNetherlands) and densitometric analyses were carried out withthe image processing program ImageJ (http://rsb.info.nih.gov/ij/).

Statistical analysis

Statistical calculations were performed using SPSS for windows(version 16.0.1, Chicago, IL, USA). Visceromotor response datawithin treatment groups were always compared with the previouspoint in time (e.g. response at day 0 with day 1, day 1 with day 5etc.). Data were analyzed with the Wilcoxon signed ranks testwhich was applied for the AUC of the relative response (normal-ized data) to colonic distention and for individual distentionvolumes. Statistical differences in Western-blot evaluations wereassessed using Mann–Whitney test. P values < 0.05 were consid-ered statistically significant in all tests.

RESULTS

Acute-stress induced persistent mast celldependent visceral hypersensitivity

We investigated whether WA-induced visceral hyper-

sensitivity is long-lasting and can be reversed by mast

cell stabilization. In maternally separated rats, WA

induced a significantly enhanced response to disten-

tion at day 1 (increased AUC in Fig. 2A,B) which

–30min

+1day

+6h

+15days

+5days

+16days

+18days

+23days

+30days

+31days

–1day

X X X X XXA Doxantrazole X X X X XXB Pre-WA α-helical CRF XX XC Post-WA α-helical CRF XXXX

X Colorectal distensions & EMG measurements=

Pharmacological intervention orvehicle treatment (i.p.)

= Sacrifice=

1 h water avoidance

Figure 1 Schematic representation of pharmacological intervention protocols. Detailed description in Material and Methods paragraph on

‘Experimental design of pharmacological intervention studies’ (A–C).

R. M. van den Wijngaard et al. Neurogastroenterology and Motility


remained elevated at post-WA days 5, 18, and 30.

Doxantrazole (Fig. 2B) administered on day 30, but not

vehicle alone (Fig. 2A) reversed the observed hypersen-

sitivity. In non-handled rats WA induced a slight, but

significant increase in sensitivity in one group only

(Fig. 2D), which was resolved on day 5 post WA.

Vehicle (Fig. 2C) and doxantrazole (Fig. 2D) treatment

on day 30 did not lead to significant changes in non-

handled rats. Supplementary Figure S1 depicts the

same set of data, but now given as relative response

to distention. Statistic evaluation indicated enhanced

post-WA response (pre-WA vs day 1) in maternally

separated rats for all three distention volumes (Supple-

mentary Figure S1A,B). A significantly changed

response (for all three volumes) was also observed

when maternally separated rats were treated with

doxantrazole between day 30 and day 31.

Application of a-helical-CRF (9-41) in maternallyseparated rats: prevention vs reversal ofstress-induced visceral hypersensitivity

A single 1 h WA-stress leads to long-term (at least

30 days) post-WA visceral hypersensitivity, suggesting

that prolonged post-WA mast cell activation may

depend on factors other than CRF and, consequently,

CRF-receptor antagonism may not suffice to reverse

existing visceral hypersensitivity. We assessed possible

differences between antagonist-driven prevention and

reversal by, respectively, administering the CRF

receptor-antagonist a-helical-CRF (9-41) in maternally

separated rats during the acute (i.e. pre-WA adminis-

tration) and chronic (i.e. post-WA administration)

hypersensitivity phase.

Sensitivity to colonic distention Antagonist-treatment

did not lead to changes in compliance (as assessed by

pressure–volume curves, data not shown). In mater-

nally separated rats, pre-WA administration of vehicle

alone led to increased AUC at 6 h and 23 days post-WA

(Fig. 3A). In contrast, pre-WA administration of

a-helical-CRF (9-41) prevented stress-induced hyper-

sensitivity to distention at the 6 h time point and AUC

remained low 23 days post-WA (Fig. 3B). Results from

the post-WA treatment groups showed WA-induced

increase in AUC at day 15. Administration of vehicle

alone (Fig. 3C) or a-helical-CRF (9-41) (Fig. 3D)

between measurements at day 15 and 16 was unable to

reverse the observed increase in visceral sensitivity.

Statistic evaluation of the relative response data

(Supplementary Figure S2) showed enhanced post-WA

response for all three distention volumes when rats

were pretreated with vehicle alone (pre-WA vs 6 h,

Figure S2A) but not upon pretreatment with a-helical-

CRF (9-41) (Figure S2B). In the post-WA treatment

groups (Figure S2C,D), WA induced enhanced response

to distention for all volumes except 2 mL in the

a-helical-CRF (9-41) treatment group (Figure S2D).

A B

C D

Figure 2 Post-WA hypersensitivity to

distention in maternally separated rats is

long-lasting and can be reversed by mast cell

stabilization. In maternally separated rats

(panels A and B), WA induced long-lasting

(30 days) hypersensitivity to distention

which was reversed by i.p. doxantrazole

treatment (gray bar, B), but not by vehicle

alone (hatched bar, A). Except for

temporarily enhanced post-WA sensitivity

on day 1 (panel D), we observed no sensi-

tivity changes in non-handled rats (panels C

and D). Data are shown as average AUC ±

SEM. Significant differences: *P < 0.05 and

**P < 0.01. AUC, area-under-curve; WA,

water avoidance.



Importantly, a-helical-CRF (9-41) treatment was

unable to reverse (day 15 vs day 16) increased post-

WA response for any of the three investigated disten-

tion volumes.

RMCP-2 expression in distal colon The combined

doxantrazole and CRF receptor-antagonist data suggest

that pre-WA a-helical-CRF (9-41) administration pre-

vented mast cell activation whereas activation was

unaffected in the post-WA treatment protocol. We

evaluated total RMCP-2 expression in stripped and

homogenized mucosa of distal colon. Protein expres-

sion was assessed by densitometric analysis of RMCP-

2/GAPDH as analyzed on Western blots (original

RMCP-2 Western blots in top panels of Fig. 4E). Rats

pretreated with a-helical-CRF (9-41) showed higher

relative RMCP-2 tissue levels than those pretreated

with vehicle alone (Fig. 4A). In contrast, in the post-

WA treatment protocol, colonic RMCP-2 levels did not

differ between a-helical-CRF (9-41) and vehicle treat-

ment groups (Fig. 4B) indicating that CRF-receptor

antagonism affects mast cell activation to a lesser

extend in this setting.

Occludin expression in distal colon When maternally

separated rats were pretreated with a-helical-CRF

(9-41), average post-WA occludin levels in colonic

mucosa were higher than those of vehicle pretreated

rats (Fig. 4C). Such differences were not observed in the

post-WA treatment protocol (Fig. 4D). Original Wes-

tern blots in bottom panels of Fig. 4E.

DISCUSSION

Prestress administration of CRF-receptor antagonists

was previously shown to prevent mast cell degranula-

tion and subsequent barrier dysfunction and develop-

ment of visceral hypersensitivity in animal

models.15,24–26 Our study confirms that the receptor

antagonist a-helical-CRF (9-41) potently prevents the

development of visceral hypersensitivity when admin-

istered before acute stress in adult predisposed rats. In

contrast, established post-WA hypersensitivity could

not be reversed by this antagonist although hypersen-

sitivity was reversed by mast cell stabilization. These

results indicate that factors other than CRF may

contribute to sustained poststress mast cell activation.

Early life stressors are known to contribute to IBS in

adults27,28 and the maternal separation model in rats is

often used to mimic such predisposing factor.10 In this

model, adverse early life experience predisposes for

complaints like visceral hypersensitivity and barrier

dysfunction later in life20,22,23,29 and these features are

also important in IBS.2,3,7–9 In contrast to others who

reported differences in baseline responsiveness to

distention between non-handled and maternally sepa-

rated rats22,29 our adult separated Long Evans rats need

an acute WA stress to bring out the hypersensitive

A

C D

B

Figure 3 Pre-WA a-helical-CRF (9-41) admi-

nistration prevents, but post-WA adminis-

tration does not reverse, hypersensitivity to

distention. Pre-WA administration (panel A)

of vehicle alone (i.p.) lead to increased AUC

at 6 h post-WA whereas administration of

a-helical-CRF (9-41) inhibited stress-induced

hypersensitivity to distention (panel B).

Panels C and D show increased post-WA

AUC (T = 15 days) in both groups. This

response was not reversed by post-WA

treatment (between days 15 and 16) with

vehicle alone (panel C) or antagonist (panel

D). Data are shown as average AUC ± SEM.

Significant differences: *P < 0.05. AUC,

area-under-curve; CRF, corticotropin-

releasing-factor; WA, water avoidance.



phenotype.20 This discrepancy may be explained by the

use of different rat strains and by our use of radiote-

lemetry for EMG measurements. This technique

allows measurement of baseline sensitivity while rats

are freely moving in their cage, herewith minimizing

unwanted stress that is induced by the measurement

procedure itself. As others often use restraint during

measurements, their baseline response may be incor-

rect because it will, unwontedly, reflect restraint stress

induced hypersensitivity to distention. We regard the

need for stress in our model a positive feature because

also in IBS acute stress is a known trigger for visceral

hypersensitivity.2 In adult maternally separated Long

Evans rats, we earlier showed an essential role for

stress-induced mast cell degranulation in the acute

phase. Pre-WA treatment with the broadly used muco-

sal mast cell stabilizer doxantrazole15,24–26,29,30 pre-

vented elicitation of colonic hypersensitivity to

distention.19 We now extended this observation to

long lasting post-WA visceral hypersensitivity: doxant-

razole treatment at post-WA day 30 was able to reverse

hypersensitivity established earlier. Most other studies

focussed on stress-induced mast cell activation in the

acute phase and never evaluated possible long-term

phenotypical consequences of acute stress. However,

chronic mast cell activation was shown to play a role

in maternally separated Wistar rats that also display an

IBS-like phenotype (barrier dysfunction and visceral

hypersensitivity).24,29 However, in contrast to Long

Evans rats, this rat strain does not require additional

acute stress at adult age to induce this mast cell

dependent phenotype in maternally separated animals.

A B

C

E

D

Figure 4 Pre- but not post-WA a-helical-CRF (9-41) administration leads to differences in mucosal RMCP-2 and occludin protein levels. Distal

colonic mucosa collected from the pre- and post-WA a-helical-CRF (9-41) treatment groups was evaluated by Western blotting. Protein expression

levels (relative to GAPDH) were quantified by densitometry. Compared with vehicle alone, antagonist pretreated maternally separated rats display

higher RMCP-2 (panel A) and occludin (panel C) protein levels. Comparison of post-WA treatment groups (vehicle vs antagonist) does not show

differences in expression of RMCP-2 (panel B) and occludin (panel D). Significant differences: *P < 0.05 and **P < 0.01. Original Western blots in

panel E. AUC, area-under-curve; CRF, corticotropin-releasing-factor; WA, water avoidance.



Accordingly, it remains unclear whether the observed

long term effects are specific for Long Evans rats or also

apply to other rat strains.

Our pre-WA CRF receptor-antagonist data confirm

earlier studies which indicated that, upon acute stress,

initial mucosal mast cell degranulation is triggered by

peripheral CRF.15,18,24–26 CRF interacts with two

receptors, CRF1- and CRF2, but with highest affinity

to CRF1.14 In brain, CRF/CFR1 was shown to be the

most relevant interaction for stress-related alterations

in colonic function. In contrast, functional studies on

human intestinal mast cells indicated that both recep-

tors are expressed- and relevant for their CRF-induced

activation.18 Similarly, in rat mast cell studies the use

of selective receptor antagonists for CRF124 and CRF2

16

implicated both receptors. On the other hand, the non-

selective receptor antagonist a-helical CRF (9-41) was

shown to preferentially block CRF2-receptors31,32 and

several groups successfully used this compound to

inhibit stress-induced mast cell degranulation and

subsequent changes in intestinal phenotype in both

rat15,24–26 and human.18 When administered peripher-

ally, this particular antagonist has poor penetration

into brain14 thus ruling out the modulation of central

CRF-signaling pathways as much as possible. In our

experiments intraperitoneal pre-WA a-helical CRF

(9-41) administration blocked mast cell dependent

visceral hypersensitivity but, despite the observation

that mast cell stabilization reversed post-WA effects,

failed to counteract chronic post-WA hypersensitivity.

These results suggest that chronic post-WA visceral

hypersensitivity involves alternative mast cell

dependent mechanisms that are less dependent on

CRF-receptor activation.

We observed earlier enhanced in situ RMCP2

expression in colonic mucosal mast cells of MS rats

which decreased to normal non-handled-level upon

WA.19 These results are corroborated by the present

Western blot quantitations of stripped colonic mucosa.

Compared with vehicle pretreated MS-rats, a-helical

CRF (9-41) pretreated animals displayed higher post-

WA RMCP2 protein-expression. This most likely

reflects RMCP-2 being retained in mast cells. In

contrast, no difference was observed when post-WA

treated rats were compared (vehicle vs antagonist),

suggesting that RMCP-2 release was equal in these

treatment groups. RMCP2 is a chymase analog and it is

known that chymase can degrade the tight junction

protein occludin.33 This may be relevant because loss

of occludin induces barrier dysfunction34 and protease-

induced occludin degradation was suggested to play a

role in IBS.6 Our occludin quantitations are in line

with the observed RMCP2 expression levels: a signif-

icant difference only occurs when rats are pretreated

with a-helical CRF (9-41). Although we have not

performed extensive barrier studies these data suggest

that a-helical CRF (9-41) may prevent stress-induced

mast cell degranulation and subsequent barrier dys-

function, but is unable to reverse it.

A possible limitation of this study could be the

timing of the post-WA a-helical CRF (9-41) adminis-

trations. However, it was shown earlier that a-helical

CRF (9-41) is not only capable of preventing CRF-

mediated effects in rat paw skin, but was also able to

reverse such effects within minutes after antagonist

application.35 The immediateness of this event sug-

gests that our protocol, three times i.p. administration

in a 24 h timeframe (each dose equal to the successful

pre-WA protocol) and the last dose given 30 min before

post-treatment distentions, should suffice to antago-

nize CRF-receptor mediated mast cell degranulation.

Another concern may be that a-helical CRF (9-41) is

mainly a CRF2-receptor antagonist and we did not

apply specific CRF1-antagonists. However, in this

article we focus on the role of mast cells in relation

to stress-induced visceral hypersensitivity and several

earlier studies showed that a-helical CRF (9-41) is

capable of inhibiting CRF-induced mast cell degranu-

lation.15,19,24–26,29,30 Furthermore, in a model of

repeated WA-exposure (10 consecutive days, 1 h day)1)

it was shown that (i) mast cells are relevant for chronic

stress induced barrier dysfunction36 and (ii) that daily

pre-WA a-helical CRF (9-41) treatment can prevent

mast cell dependent antigen uptake.37 The same model

was also used to investigate the development of

visceral hypersensitivity which was prevented by daily

pre-WA administration (subcutaneously) of the BBB-

crossing CRF1-receptor antagonist CP-154,526.38 This

confirmed earlier observations that central CRF1-

receptors are essential in the acute peri-stress time

frame. However, reversal of hypersensitivity by post

stress CP-154,526 administration at day 11 alone was

only partially successful. The combined above data

suggest that ongoing post-stress hypersensitivity to

distention may, at least to some degree, be due to non-

CRF-receptor dependent triggers.

Unlike rats that are exposed to 1 h of water avoidance

only, IBS-patients most likely experience repeated stress

episodes that are considered as chronic stress. Although

this suggests a continuous role for CRF in patients, our

data support the outcome of two recent clinical trials in

which CRF-receptor antagonists showed no patient

benefit.39,40 Together with the observation that mast

cell stabilization may be an effective treatment in IBS,13

these trial results support our proposed mechanism

that, following initial stress-induced mast cell



activation by CRF, other mast cell triggers become

relevant as well. As stress-induced barrier dysfunction

is known to be associated with influx of luminal

bacteria/antigens which can lead to antigen specific

immunity,36,37 we expect humoral immune responses

to be involved. In this respect, it was shown that in

maternal separated Sprague-Dawley rats WA at adult

age induced increased transepithelial transport of mac-

romolecules which could be blocked by a-helical CRF

(9-41) pretreatment.23 Using the same rat strain, it was

later shown that exposure to maternal separation

induces bacterial adherence to- and penetration into

colonic epithelium during and shortly after the separa-

tion period.41 Culture of washed and homogenized

segments of distal colon confirmed increased bacterial

presence in colonic-wall of maternally separated rats

and penetration was accompanied by increased translo-

cation to the spleen. These data show that, in the

maternal separation model, early neonatal mucosal

antigen-exposure can facilitate antigen-priming of the

humoral immune response. At adult age subsequent

CRF-induced barrier dysfunction can challenge this

response, which may explain how mast cells can then,

after challenge, be relevant without further role of CRF.5

Pilot experiments performed in our own laboratory

confirm that humoral immune responses may have a

role in the maternal separation model42 and recent

observations of increased anti-flagellin antibody titers

suggest that humoral mechanisms may also be relevant

to IBS.43

In summary, our investigations were performed in

the rat model of maternal separation in which acute

stress induces, similar to IBS, visceral hypersensitivity.

Although the stressor used was 1 h of WA only, the

observed hypersensitivity to distention lasted for at

least 1 month and was mast cell dependent. The CRF-

receptor antagonist a-helical CRF (9-41) could prevent,

but not reverse this stress-induced hypersensitivity. If

these results also apply to IBS, they suggest that

antagonizing CRF receptors alone will not be sufficient

for the reversal of stress-induced and mast cell depen-

dent complaints in this disorder.

ACKNOWLEDGMENTS

These investigations were supported by The Netherlands TopInstitute Pharma (TIP), grant number T1-215-1 and by funding forthe IPODD consortium under Grant Agreement 202020 of theSeventh Research Framework Programme of the European Union.GEB was supported by a grant (Odysseus program, G.0905.07) ofthe Flemish ‘‘Fonds Wetenschappelijk Onderzoek’’ (FWO).

AUTHOR CONTRIBUTIONS

RvdW participated in the study concept and design, study

supervision, analysis and interpretation of data, and drafting of

the manuscript; OIS participated in acquisition, analysis and

interpretation of data, statistical analysis, and drafting of the

manuscript; SAvD and OW participated in acquisition, analysis,

and interpretation of data; MMW and WJdJ discussed the results

and implications and commented on the manuscript at all stages;

GEB participated in the study concept and design, study supervi-

sion, interpretation of data, and critical revision of the manuscript

for important intellectual content.

COMPETING INTERESTS

The authors have no competing interests.

REFERENCES

1 Longstreth GF, Thompson WG, CheyWD, Houghton LA, Mearin F, SpillerRC. Functional bowel disorders.Gastroenterology 2006; 130: 1480–91.

2 Posserud I, Agerforz P, Ekman R,Bjornsson ES, Abrahamsson H, Sim-ren M. Altered visceral perceptualand neuroendocrine response inpatients with irritable bowelsyndrome during mental stress. Gut

2004; 53: 1102–8.3 Posserud I, Syrous A, Lindstrom L,

Tack J, Abrahamsson H, Simren M.Altered rectal perception in irritablebowel syndrome is associated withsymptom severity. Gastroenterology

2007; 133: 1113–23.

4 Gareau MG, Silva MA, Perdue MH.Pathophysiological mechanisms ofstress-induced intestinal damage.Curr Mol Med 2008; 8: 274–81.

5 Van Den Wijngaard RM, Klooker TK,De Jonge WJ, Boeckxstaens GE.Peripheral relays in stress-inducedactivation of visceral afferents in thegut. Auton Neurosci 2010; 153: 99–105.

6 Coeffier M, Gloro R, Boukhettala Net al. Increased proteasome-mediateddegradation of occludin in irritablebowel syndrome. Am J Gastroenterol

2010; 105: 1181–8.7 Dunlop SP, Hebden J, Campbell E

et al. Abnormal intestinal perme-ability in subgroups of diarrhea-predominant irritable bowelsyndromes. Am J Gastroenterol 2006;101: 1288–94.

8 Piche T, Barbara G, Aubert P et al.

Impaired intestinal barrier integrityin the colon of patients with irritablebowel syndrome: involvement of sol-uble mediators. Gut 2009; 58: 196–201.

9 Spiller RC, Jenkins D, Thornley JPet al. Increased rectal mucosal ente-roendocrine cells, T lymphocytes,and increased gut permeabilityfollowing acute Campylobacter

enteritis and in post-dysentericirritable bowel syndrome. Gut 2000;47: 804–11.

10 Barreau F, Ferrier L, Fioramonti J,Bueno L. New insights in the etiologyand pathophysiology of irritablebowel syndrome: contribution ofneonatal stress models. Pediatr Res

2007; 62: 240–5.



11 Barbara G, Wang B, Stanghellini Vet al. Mast cell-dependent excitationof visceral-nociceptive sensory neu-rons in irritable bowel syndrome.Gastroenterology 2007; 132: 26–37.

12 Buhner S, Li Q, Vignali S et al. Acti-vation of human enteric neurons bysupernatants of colonic biopsy speci-mens from patients with irritablebowel syndrome. Gastroenterology

2009; 137: 1425–34.13 Klooker TK, Braak B, Koopman KE

et al. The mast cell stabiliser ketoti-fen decreases visceral hypersen-sitivity and improves intestinalsymptoms in patients with irritablebowel syndrome. Gut 2010; 59: 1213–21.

14 Stengel A, Tache Y. Neuroendocrinecontrol of the gut during stress: cor-ticotropin-releasing factor signalingpathways in the spotlight. Annu Rev

Physiol 2009; 71: 219–39.15 Gue M, Del Rio-Lacheze C, Eutam-

ene H, Theodorou V, Fioramonti J,Bueno L. Stress-induced visceralhypersensitivity to rectal distensionin rats: role of CRF and mast cells.Neurogastroenterol Motil 1997; 9:271–9.

16 Teitelbaum AA, Gareau MG, Jury J,Yang PC, Perdue MH. Chronicperipheral administration of cortico-tropin-releasing factor causes colonicbarrier dysfunction similar to psy-chological stress. Am J Physiol Gas-

trointest Liver Physiol 2008; 295:G452–9.

17 Santos J, Yates D, Guilarte M, VicarioM, Alonso C, Perdue MH. Stressneuropeptides evoke epithelialresponses via mast cell activation inthe rat colon. Psychoneuroendocri-

nology 2008; 33: 1248–56.18 Wallon C, Yang PC, Keita AV et al.

Corticotropin-releasing hormone(CRH) regulates macromolecular per-meability via mast cells in normalhuman colonic biopsies in vitro. Gut2008; 57: 50–8.

19 Van Den Wijngaard RM, Klooker TK,Welting O et al. Essential role forTRPV1 in stress-induced (mast cell-dependent) colonic hypersensitivityin maternally separated rats. Neuro-

gastroenterol Motil 2009; 21: 1107–e94.

20 Welting O, Van Den Wijngaard RM,De Jonge WJ, Holman R, Boeckxsta-ens GE. Assessment of visceralsensitivity using radio telemetry in arat model of maternal separation.

Neurogastroenterol Motil 2005; 17:838–45.

21 Van Den Wijngaard RM, Welting O,Bulmer DC et al. Possible role forTRPV1 in neomycin-induced inhibi-tion of visceral hypersensitivity inrat. Neurogastroenterol Motil 2009;21: 863–e60.

22 Coutinho SV, Plotsky PM, Sablad Met al. Neonatal maternal separationalters stress-induced responses toviscerosomatic nociceptive stimuli inrat. Am J Physiol Gastrointest Liver

Physiol 2002; 282: G307–16.23 Soderholm JD, Yates DA, Gareau

MG, Yang PC, MacQueen G, PerdueMH. Neonatal maternal separationpredisposes adult rats to colonic bar-rier dysfunction in response to mildstress. Am J Physiol Gastrointest Li-

ver Physiol 2002; 283: G1257–63.24 Barreau F, Cartier C, Leveque M et al.

Pathways involved in gut mucosalbarrier dysfunction induced in adultrats by maternal deprivation: corti-cotrophin-releasing factor and nervegrowth factor interplay. J Physiol2007; 580: 347–56.

25 Keita AV, Soderholm JD, Ericson AC.Stress-induced barrier disruption ofrat follicle-associated epithelium in-volves corticotropin-releasing hor-mone, acetylcholine, substance P,and mast cells. NeurogastroenterolMotil 2010; 22: 770–2.

26 Santos J, Saunders PR, Hanssen NPet al. Corticotropin-releasing hor-mone mimics stress-induced colonicepithelial pathophysiology in the rat.Am J Physiol 1999; 277: G391–9.

27 Chitkara DK, van Tilburg MA, Blois-Martin N, Whitehead WE. Early liferisk factors that contribute to irritablebowel syndrome in adults: a system-atic review. Am J Gastroenterol 2008;103: 765–74.

28 Klooker TK, Braak B, Painter RC et al.

Exposure to severe wartime condi-tions in early life is associated withan increased risk of irritable bowelsyndrome: a population-based cohortstudy. Am J Gastroenterol 2009; 104:2250–6.

29 Barreau F, Cartier C, Ferrier L, Fiora-monti J, Bueno L. Nerve growth factormediates alterations of colonic sensi-tivity and mucosal barrier induced byneonatal stress in rats. Gastroenter-

ology 2004; 127: 524–34.30 Stein J, Ries J, Barrett KE. Disruption

of intestinal barrier function asso-ciated with experimental colitis:

possible role of mast cells. Am JPhysiol 1998; 274: G203–9.

31 Rivier J, Gulyas J, Kirby D et al.

Potent and long-acting corticotropinreleasing factor (CRF) receptor 2selective peptide competitive antago-nists. J Med Chem 2002; 45: 4737–47.

32 Smart D, Coppell A, Rossant C, HallM, McKnight AT. Characterisationusing microphysiometry of CRFreceptor pharmacology. Eur J Phar-macol 1999; 379: 229–35.

33 Ebihara N, Funaki T, Murakami A,Takai S, Miyazaki M. Mast cellchymase decreases the barrier func-tion and inhibits the migration ofcorneal epithelial cells. Curr Eye Res

2005; 30: 1061–9.34 Al-Sadi R, Khatib K, Guo S, Ye D,

Youssef M, Ma T. Occludin regulatesmacromolecule flux across theintestinal epithelial tight junctionbarrier. Am J Physiol Gastrointest

Liver Physiol 2011; 300: G1054–64.

35 Wei ET, Serda S, Tian JQ. Protectiveactions of corticotropin-releasing fac-tor on thermal injury to rat pawskin.J Pharmacol Exp Ther 1988; 247:1082–5.

36 Soderholm JD, Yang PC, Ceponis P et

al. Chronic stress induces mast cell-dependent bacterial adherence andinitiates mucosal inflammation in ratintestine. Gastroenterology 2002;123: 1099–108.

37 Yang PC, Jury J, Soderholm JD, Sher-man PM, McKay DM, Perdue MH.Chronic psychological stress in ratsinduces intestinal sensitization toluminal antigens. Am J Pathol 2006;168: 104–14.

38 Larauche M, Bradesi S, Million M et

al. Corticotropin-releasing factor type1 receptors mediate the visceralhyperalgesia induced by repeatedpsychological stress in rats. Am J

Physiol Gastrointest Liver Physiol2008; 294: G1033–40.

39 Dukes GE, Mayer EA, Kelleher DL,Hicks KJ, Boardley RL, Alpers DH.A randomised double blind, placebocontrolled, crossover study to evalu-ate the efficacy and safety of thecorticotrophin releasing factor 1(CRF1) receptor antagonist GW876008in IBS patients. Neurogastroenterol

Motil 2009; 21(Suppl.): 84.40 Sweetser S, Camilleri M, Linker Nord

SJ et al. Do corticotropin releasingfactor-1 receptors influence colonic



transit and bowel function in womenwith irritable bowel syndrome? Am J

Physiol Gastrointest Liver Physiol

2009; 296: G1299–306.41 Gareau MG, Jury J, Yang PC, Mac-

Queen G, Perdue MH. Neonatalmaternal separation causes colonicdysfunction in rat pups including

impaired host resistance. Pediatr Res2006; 59: 83–8.

42 van Diest SA, Stanisor OI, Welting Oet al. Free immunoglobulin lightchains may be involved in stress-induced visceral hypersensitivity inmaternal separated rats. Gastroenter-

ology 2010; 138: S-36.

43 Schoepfer AM, Schaffer T,Seibold-Schmid B, Muller S, SeiboldF. Antibodies to flagellin indicatereactivity to bacterial antigens in IBSpatients. Neurogastroenterol Motil2008; 20: 1110–8.

SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article:

Figure S1. Vehicle or doxantrazole treatment between day 30 and 31. Data correspond to those of Fig. 2, but are

depicted in a different fashion. Left side: relative response to distention. Right side: statistics for individual volumes.

(A) vehicle treated maternally separated rats, (B) doxantrazole treated maternally separated rats, (C) vehicle treated

non-handled rats and (D) doxantrazole treated non-handled rats. WA induces enhanced sensitivity to distention in

maternally separated, but not in non-handled rats (pre-WA vs day 1). Hypersensitivity in maternally separated rats is

reversed by doxantrazole, but not by vehicle treatment (day 30 vs day 31).

Figure S2. Pre-WA and post-WA treatment with vehicle or a-helical-CRF (9-41).Data correspond to those of Fig. 3,

but are depicted in a different fashion. Left side: relative response to distention. Right side: statistics for individual

volumes. All four panels concern maternally separated rats: (A) pre-WA treatment with vehicle alone, (B) pre-WA

treatment with a-helical-CRF (9-41), (C) post-WA treatment with vehicle alone and (D) post-WA treatment with

a-helical-CRF (9-41). a-helical-CRF (9-41) can prevent, but not reverse post-WA visceral hypersensitivity.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials

supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for

the article.


� 2011 Blackwell Publishing Ltd e111

Peripheral α-helical CRF (9-41) does not reverse stress-induced mast cell dependent visceral...

Documents

Transcript of Peripheral α-helical CRF (9-41) does not reverse stress-induced mast cell dependent visceral...