Stress neuropeptides evoke epithelial responsesvia mast cell activation in the rat colon

Javier Santos a,*, Derrick Yates b, Mar Guilarte a, Maria Vicario a,Carmen Alonso a, Mary H. Perdue b

Psychoneuroendocrinology (2008) 33, 1248—1256

ava i lab le at www.sc ienced i rect .com

journa l homepage: www.el sev ier.com/locate/psyneuen

aDigestive Diseases Research Unit, Institut de Recerca Vall d’Hebron, Department of Gastroenterology, Hospital Universitari Valld’Hebron, Universitat Autonoma de Barcelona, Department of Medicine, Barcelona, Spainb Intestinal Disease Research Programme, Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMasterUniversity, Hamilton, Ontario L8N 3Z5, Canada

Received 9 January 2008; received in revised form 11 June 2008; accepted 1 July 2008

KEYWORDSCorticotropin-releasingfactor;Sauvagine;Mast cells;Permeability;Secretion;Rat colon

Summary

Background: Previously, we showed that corticotropin-releasing factor (CRF) injected i.p.mimicked epithelial responses to stress, both stimulating ion secretion and enhancing perme-ability in the rat colon, and mast cells were involved. However, the ability of CRF-sensitivemucosal/submucosal loops to regulate intestinal barrier and the participation of resident mastcells are unclear.Methods: We examined colonic epithelial responses to stress-like peptides in Wistar—Kyoto(WKY), and mast cell-deficient (Ws/Ws) and their +/+ littermate control rats in distal segmentsmounted in Ussing chambers. Short-circuit current (ion secretion), flux of horseradish peroxidase(macromolecular permeability), and the release of rat mast cell protease II were measured inresponse to CRF [10�6 to 10�8 M] or sauvagine [10�8 to 10�10 M] in tissues pretreated withastressin, doxantrazole, or vehicle.Results: Stress-like peptides (sauvagine > CRF) induced a dose-dependent increase in short-circuit current (maximal at 30 min), and significantly enhanced horseradish peroxidase flux andprotease II release in WKY. Epithelial responses were inhibited by both astressin and doxantra-zole, and significantly reduced in tissues from Ws/Ws rats.Conclusion: The stress mediators CRF and sauvagine modulate barrier function in the rat colonacting onmucosal/submucosal CRF receptor-bearing cells, throughmast cell-dependent pathways.# 2008 Elsevier Ltd. All rights reserved.

Abbreviations: CRF, corticotropin-releasing factor; HRP, horse-radish peroxidase; Isc, short-circuit current; NS, not significant; RMCPII, rat mast cell protease II; SVG, sauvagine; WKY, Wistar—Kyoto.* Corresponding author at: Digestive Diseases Research Unit, Insti-

tut de Recerca Vall d’Hebron, Department of Gastroenterology,Hospital Universitari Vall d’Hebron, Universitat Autonoma de Barce-lona, Barcelona, Spain. Fax: +34 934894032.

E-mail address: jsantos@ir.vhebron.net (J. Santos).

0306-4530/$ — see front matter # 2008 Elsevier Ltd. All rights reservedoi:10.1016/j.psyneuen.2008.07.002

1. Introduction

A number of studies in humans and experimental animals nowindicate that stress is associated with abnormalities in visc-eral perception (Grundy et al., 2006; Liebregts et al., 2007),motility (Tache and Bonaz, 2007), and epithelial function(Barclay and Turnberg, 1987; Alonso et al., 2008) in the small

d.

Stress-mast cell intraepithelial networks regulate colonic barrier 1249

and large bowel. These findings may help to explain theproposed role of stress in the modulation of mucosal inflam-mation and its putative participation in physiopathologicalevents underlying common gastrointestinal disorders such asirritable bowel syndrome (Bennett et al., 1998) and inflam-matory bowel disease (Mawdsley and Rampton, 2005). How-ever, the pathways and molecular mechanisms by whichstress induces intestinal dysfunction remain unclear.

The classical view of the intestinal epithelium as a merephysical barrier between the luminal content and the inter-nal milieu also involved in digestion and absorption of nutri-ents has been overtaken by its increasingly recognized role asan active player in the modulation of local inflammatoryevents and gut functional homeostasis (Fiocchi, 1997; San-sonetti, 2004). Growing and compelling evidence shows thatstress regulates epithelial function. Corticotropin-releasingfactor (CRF), a 41-amino acid peptide, and related analogueslike the amphibian 40-amino acid peptide sauvagine (SVG),mediate endocrine/immune, autonomic, visceral and beha-vioral responses to stress acting on high affinity membrane-bound receptors on target cells (Bale and Vale, 2004). Twomain receptor subtypes, CRF1 and CRF2, have been charac-terized in mammals (Turnbull and Rivier, 1997) but while CRFand SVG have similar affinity for CRF1, SVG displays higheraffinity for CRF2 than CRF (Grigoriadis et al., 1996). Othersand we have shown that stress stimulates epithelial ionsecretion and permeability (Santos et al., 1998; Barreauet al., 2004; Alonso et al., 2008) and modulates mucosalimmune and inflammatory responses (Soderholm et al.,2002a; Yang et al., 2006) in human and rat small intestineand colon. Moreover, peripheral CRF provoked transport andbarrier abnormalities resembling those observed in the intes-tine of stressed rats (Santos et al., 1999), effects that wereinhibited by the non-selective CRF antagonist, a-helicalCRF9-41 (Saunders et al., 2002; Soderholm et al., 2002b).

Mast cells regulate barrier physiology in normal as well asinflamed intestine, in rats and humans (Crowe et al., 1997;Berin et al., 1998). Furthermore, they play a key role in stress-mediated intestinal epithelial responses, including ion secre-tion, transport of macromolecules andmucin release, possiblyviaCRF-activated pathways (Castagliuolo et al., 1996; Barreauet al., 2008). Despite convincing evidence of intestinal expres-sion of CRF-like peptides and receptors and its production bylocal immunocytes and enteroendocrine cells (Chatzaki et al.,2004; Tache and Bonaz, 2007), only recently a role for sub-epithelial CRF-mast cell loops in the regulation of colonicpermeability in human biopsies has been reported (Wallonet al., 2008). However, the contribution of mucosal/submu-cosal CRF-based autocrine/paracrine networks and the invol-vement of residentmast cells in the control of barrier functionin the rat intestine are still unresolved.

The aims of the present study were to examine the abilityof CRF-sensitive mucosal/submucosal circuits to regulateepithelial physiology in the rat colon, and to determinethe participation of resident mast cells in these responses.

2. Methods

2.1. Animals

Experiments were performed on tissues from male Wistar—Kyoto (WKY) rats (200—250 g; Charles River Laboratories, St.

Constant, QC, Canada). Some studies involved the use ofmast cell-deficient Ws/Ws rats and their normal +/+ litter-mate controls (250—300 g; colony at McMaster University).Ws/Ws rats were obtained by breeding male and female Ws/+heterozygous rats from the original colony developed by Y.Kitamura, Osaka University, Japan (Niwa et al., 1991). Ws/Wsrats have a 12-base deletion in the tyrosine kinase domain ofthe c-kit gene (Tsujimura et al., 1991) that results in theabsence of mast cells andmelanocytes and a reduced numberof erythrocytes. By ten weeks of age erythrocyte numbers aregreatly recovered in Ws/Ws rats (Niwa et al., 1991), whichalso show food intake and growth curves similar to +/+ rats(Santos et al., 2000) but still lack intestinal mast cells, while+/+ rats have the normal numbers of entirely functional mastcells (Berin et al., 1998). Rats, housed two per cage, weremaintained on a normal 12:12-h dark/light cycle and pro-vided with food and water ad libitum. The Animal CareCommittee at McMaster University approved all procedures.

2.2. Epithelial measurements in Ussingchambers

After twoweeks of daily handling by the same investigator (toavoid inadvertent stress from human contact), rats wereeuthanized by decapitation. The distal colon was removed,placed in 37 8C oxygenated Krebs, stripped of longitudinalmuscle and myenteric plexus and opened along the mesen-teric border. Four adjacent pieces from each rat weremounted in Ussing chambers (World Precision Instruments,Sarasota, FL). The chamber opening exposed 0.6 cm2 oftissue surface area to 8 ml of circulating oxygenated Krebsbuffer at 37 8C. The buffer contained (in mM) 115NaCl,1.25CaCl2, 1.2MgCl2, 2.0KH2PO4, and 25NaHCO3 (pH 7.35).In addition, the serosal buffer contained 10 mM glucose as anenergy source, osmotically balanced by 10 mM mannitol inthe mucosal buffer. The chambers contained agar-saltbridges to monitor the potential difference across the tissueand to inject the required short-circuit current (Isc) to main-tain a zero potential difference as registered via an auto-mated voltage clamp (World Precision Instruments). Isc (inmA/cm2), a measure of net active ion transport, wasrecorded by a computer connected to the voltage-clampsystem. Tissue conductance, an indicator of ion permeabilityand tissue viability, was calculated according to Ohm’s lawand expressed as milli-Siemens/cm2.

Uptake of macromolecules was assessed by measuring themucosal-to-serosal flux of horseradish peroxidase (HRP). HRP(type VI, Sigma Chemical Co., St. Louis, MO) was added at10�5 M to the luminal buffer 20 min after the tissues weremounted, and allowed to equilibrate for 30 min. Serosalsamples (0.5 ml) were then obtained at 30-min intervalsfor 2 h (volume in the chamber maintained by replacing withglucose buffer). HRP activity was determined by a modifiedWorthington method as previously described (Santos et al.,1999). The mucosal-to-serosal flux of HRP was calculatedaccording to a standard formula (Saunders et al., 2002) andexpressed as picomoles per hour per square centimeter.

2.3. Drugs and treatments

Rat/human CRF (Peninsula Laboratories, Inc., Belmont, CA),‘‘skin frog’’ SVG, astressin, cyclo-(30-33)-[D-Phe12,Nle12,N-

1250 J. Santos et al.

le21,38,Glu30,Lys33] r/h CRF-(12-41) (kindly donated by Dr. J.Rivier, Salk Institute, Clayton Foundation Laboratories forPeptide Biology, La Jolla, CA), and doxantrazole (a gift fromBurroughs Wellcome Co., Research Triangle Park, NC) weredissolved following manufacturers’ instructions, aliquotedand kept frozen at �80 8C until used. Astressin displayshigh affinity for both CRF receptor subtypes and is morepotent than a-helical CRF12-41 in blocking various CRF- andSVG-mediated effects (Gulyas et al., 1995; Hillhouse andGrammatopoulos, 2006). Rat mast cell protease II (RMCP II)levels were also evaluated in some experiments to clarifythe participation of mucosal mast cells. RMCP II was mea-sured by ELISA (Moredun Sientific Ltd., Midlothian, Scot-land) in 0.5 ml buffer samples (0.25 ml serosal + 0.25 mlmucosal), as described (Vergara et al., 2002). RMPC IIconcentration was expressed in nanomoles per gram(wet weight) of tissue (Barreau et al., 2008). Bacitracin(Sigma), aprotinin (Miles Canada Inc., Etobicoke, ON),leupeptin, and phosphoramidon (Peninsula) were also usedin some studies.

2.4. Experimental design

Baseline values for Isc and conductance were calculated atequilibrium, 20 min after the tissues were mounted, andthen every 30 min for 2 h. The HRP flux was determinedover at least two stable 30-min periods after equilibration.Tissues from each rat were matched according to baselineIsc and conductance (paired tissues not differing by morethan 20%). Then, tissue pairs were exposed to the activedrug (CRF/SVG) or vehicle (sterile saline for both). Treat-ment assignment was based on a triple restricted rando-mization process: (A) One tissue pair from each rat wasrandomly allocated to receive either CRF [10�6 to 10�8 M]/vehicle (1) or SVG [10�8 to 10�10 M]/vehicle (2), using acomputerized random number generator tool with norepeats (1 or 2); the second tissue pair was then allocated,by minimization, to the remaining option; (B) the firsttissue of each pair was randomly allocated to receivethe active drug (1) or its vehicle (2), whereas the secondtissue of each pair was assigned to the remaining option,using the same methods; (C) when four tissue pairs (drug/vehicle) for each dose were available, the following tissuepairs were exposed to just different doses of active drugsselected as described in (B), until groups (n = 12—16) werecompleted.

Drugs and vehicles were administered on the serosal sideof the tissues. In preliminary experiments, an enzyme inhi-bitor cocktail, known to prevent peptide digestion in similarsystems, was added to the buffer:bacitracin [2 � 10�5 M],leupeptin [9 � 10�6 M], phosphoramidon [2 � 10�6 M], andaprotinin [500 kallikrein-inactivating units/ml]. Becauseelectrophysiological responses to the peptides were notaffected by this treatment, we did not use protease inhibitorsin subsequent experiments.

Additional tissues were exposed to astressin [10�5 to10�8 M]/vehicle (double-distilled water) or doxantrazole[10�5 M]/vehicle (NaHCO3, 5% w/v), 30 min before CRF,SVG or saline. Doses for CRF, SVG, astressin, and doxantrazolewere based on previous reports showing their ability toinduce or inhibit a variety of electrophysiological, biochem-ical and morphological responses in different in vitro pre-

parations (Kiang, 1997; Santos et al., 1999). RMCP II wasmeasured in separate experiments at equilibrium and 1hafter CRF [10�6 M], SVG [10�9 M] or saline. To ascertainthe role of mast cells, tissues from Ws/Ws and +/+ rats weretreated with the maximal effective doses of CRF and SVG.Treatment assignment for astressin, doxantrazole andWs/Wsand +/+ experiments was performed in a way similar as thatdescribed before.

2.5. Statistical analysis

Results are expressed as means � S.E.M. unless otherwisestated. For each tissue, Isc and conductance responses werecalculated by subtracting baseline values from maximumvalues after treatment, and expressed as the increment(D) for that period. For each treatment, paired statisticalcomparisons were performed. Multiple groups were com-pared by Dunnett’s test and Tukey—Kramer test following asignificant one-way analysis of variance. Single comparisonswere performed by paired or unpaired Student’s t-test whereappropriate. P < 0.05 was considered significant.

3. Results

3.1. Effect of CRF and SVG on colonic short-circuit current

All tissue groups used to evaluate drug and vehicle responsesdisplayed equal Isc and conductance baseline values(Table 1). The typical Isc response CRF and SVG showed adose-dependent gradual increase detectable within 3 min,which reached a maximum at �30 min, and decreased butremained above baseline thereafter (Fig. 1). The increase inIsc was paralleled by an increase in potential difference sothat conductance did not change significantly at any point.The response to CRF ranged from the peak average incrementabove baseline of 56% at 10�6 M (P < 0.05), to 26% at 10�7 M(P NS), and 20% at 10�8 M (P NS), as compared to 16% forvehicle (Fig. 2A). The predominant CRF2 agonist SVG (Fig. 2B)also increased Isc above baseline by 64% at 10�8 M, by 114% at10�9 M, and by 48% at 10�10 M, as compared to 12.5% forvehicle (P < 0.05 for all comparisons).

3.2. Effect of CRF and SVG on HRP flux across thecolonic mucosa

HRP flux was enhanced by CRF in a concentration-dependentmanner (Fig. 3A), ranging from a 3.9-fold increase at 10�6 M(15.2 � 2.6 pmol h�1 cm�2, P < 0.05 vs. vehicle) to non-sig-nificant increase at lower CRF concentrations, compared totissues treated with vehicle (3.9 � 0.9 pmol h�1 cm�2). SVGalso induced a marked increase in HRP flux thatpeaked at 10�9M (20.9 � 5.8; pmol h�1 cm�2; vehicle:4.4 � 1.0 pmol h�1 cm�2, P < 0.05). Higher concentrationsof SVG were not associated with further enhancement inHRP flux (Fig. 3B).

3.3. Effect of the CRF antagonist astressin onCRF-and SVG-stimulated colonic Isc and HRP flux

Astressin, when added at different concentrations to theserosal compartment, did not alter either Isc or HRP flux,

Table 1 Baseline electrophysiological characteristics of tissues used in experiments

Rats Drug Isc (mA/cm2) G (mS/cm2) n

Value Range P Value Range P

WKY CRF 39.9(14.5) 12.8—64.3 0.89 21.2(6.1) 12.0—35.2 0.54 68SVG 37.5(15.1) 10.2—64.4 22.7(6.2) 10.6—37.8 68Vehicle 37.3(15.0) 12.0—63.0 20.9(6.8) 11.5—36.6 40Astressin 36.7(14.4) 11.2—64.2 22.4(6.9) 11.5—38.6 68Vehicle 36.4(13.1) 13.5—61.4 23.3(6.8) 12.3—36.5 32DOX 38.8(13.1) 16.3—64.3 22.5(5.3) 16.5—36.8 28Vehicle 37.4(13.5) 16.6—63.3 21.5(6.8) 12.3—37.1 28

Ws/Ws CRF 34.8(9.4) 20.3—52.5 0.98 25.7(7.1) 16.7—38.5 0.85 8SVG 35.6(9.5) 19.3—48.8 25.9(7.5) 15.7—38.8 8Vehicle 35.3(8.7) 18.5—48.6 24.5(4.0) 18.5—31.5 12

+/+ CRF 35.7(10.6) 20.9—51.2 0.98 26.3(4.4) 21.3—34.8 0.70 8SVG 35.4(11.8) 18.8—52.1 24.5(5.8) 17.0—33.2 8Vehicle 36.2(8.7) 19.8—50.6 24.9(3.2) 21.0—31.6 12

Data values are expressed as mean (S.D.). No statistical differences were observed for Isc or G comparisons within rat species and treatmentsubgroups, as shown by P values after a one-way analysis of variance followed by Tukey—Kramer test. CRF, corticotropin-releasing factor;DOX, doxantrazole; G, conductance; Isc, short-circuit current; SVG, sauvagine; WKY, Wistar—Kyoto; Ws/Ws, mast cell-deficient; +/+,littermate controls for Ws/Ws.

Stress-mast cell intraepithelial networks regulate colonic barrier 1251

comparedwith its vehicle. Astressin,when administered in 10-fold excess of the agonist, abolished Isc (21.3 � 5.6 mA/cm2;5.6 � 1.2 mA/cm2, P < 0.05, vehicle vs. astressin) and HRPflux increase (14.5 � 2.9 pmol h�1 cm�2; 5.7 � 1.3 pmolh�1 cm�2, P < 0.05, vehicle vs. astressin) in response to CRF10�6 M (Fig. 4). Likewise, astressin reduced Isc (47.8 � 9.6 mA/cm2; 17.6 � 5.8 mA/cm2, P < 0.05, vehicle vs. astressin) andHRP flux increase (21.9 � 3.8 pmol h�1 cm�2; 10.4 � 1.6pmol h�1 cm�2, P < 0.05, vehicle vs. astressin) in responseto SVG 10�9 M (Fig. 4). These results indicate that epithelialresponses to CRF and SVG involve specific CRF receptor activa-tion.

Figure 1 Effect of corticotropin-releasing factor (CRF) and sauvarepresentative changes in short-circuit current (Isc) and conductancecolon of WKY rats mounted in Ussing chambers after the serosal expospeptides induced a marked and gradual increase in Isc, which peakelevated above baseline. Conductance was not affected.

3.4. Role of intestinal mast cells in epithelialresponses to CRF and SVG

Themast cell stabilizer doxantrazole at 10�5 M did not changeIsc or HRP flux compared to its vehicle. However, doxantrazoleabolished the increase in Isc (Fig. 5) to CRF at 10�6 M(20.8 � 4.3 mA/cm2; 3.1 � 1.1 mA/cm2, P < 0.05, vehicle vs.doxantrazole), and to SVG at 10�9 M (43.2 � 5.9 mA/cm2;4.7 � 2.6 mA/cm2, P < 0.05, vehicle vs. doxantrazole). Simi-larly, doxantrazole abolished HRP flux responses(Fig. 5) to CRF at 10�6 M (15.0 � 3.8 pmol h�1 cm�2;3.7 � 1.8 pmol h�1 cm�2, P< 0.05, vehicle vs. doxantrazole),

gine (SVG) on colonic mucosal electrophysiology. Tracings show(vertical pulses on top of tracings) in segments from the distal

ure to most effective doses of CRF, SVG and vehicle (saline). Bothed after 30 min and slowly decreased thereafter but remained

Figure 2 Dose—response curves for corticotropin-releasingfactor (CRF) and sauvagine (SVG) on colonic short-circuit current(Isc). Distal colonic tissues fromWKY rats were mounted in Ussingchambers. Different concentrations of CRF (upper panel, A),SVG, (lower panel, B), or vehicle were administered on theserosal side at equilibrium (baseline) and Isc recorded at baselineand 30 min later. Values are expressed as the averaged meanincrements (D) above baseline � S.E.M. for each CRF/SVG con-centration, n = 12—16 tissues/group. *P < 0.05 vs. Dvehicle (Dfor CRF vehicle: 6.62 � 2.4 mA/cm2; D for SVG vehicle:4.4 � 2.2 mA/cm2, n = 20 tissues/group).

Figure 3 Dose—response curves for corticotropin-releasingfactor (CRF) and sauvagine (SVG) on horseradish peroxidase(HRP) flux across the colonic mucosa. Distal colonic tissues fromWKY rats were mounted in Ussing chambers. Different concen-trations of CRF (upper panel, A), SVG (lower panel, B), or vehicle(0.9% saline) were added to the serosal side at equilibrium(baseline). HRP flux was calculated as the average value of 2consecutive 30-min stable flux periods. Values indicate themean � S.E.M. for each CRF/SVG concentration, n = 8—16 tis-sues/group. *P < 0.05 vs. vehicle (CRF vehicle: 3.9 � 0.9pmol h�1 cm�2; SVG vehicle: 4.4 � 1.0 pmol h�1 cm�2, n = 8—10 tissues/group).

Figure 4 Effect of astressin on corticotropin-releasing factor- and sauvagine-induced colonic epithelial responses. Distal colonictissues from WKY rats mounted in Ussing chambers were exposed to increasing concentrations of astressin administered on the serosalside, 30 min before the addition of corticotropin-releasing factor (10�6 M, black bars), sauvagine (10�9 M, gray bars) or saline (emptybars). The increase in short-circuit current (Isc) above baseline and the flux of horseradish peroxidase (HRP) were measured. Valuesindicate the mean � S.E.M., n = 8—16 tissues/group; *P < 0.05 vs. vehicle.

1252 J. Santos et al.

Figure 5 Role of intestinal mast cells on corticotropin-releasing factor- and sauvagine-induced colonic epithelial responses. Distalsegments fromWKY rats (left panels), pretreated (�30 min) with serosal doxantrazole/vehicle, as well as untreated tissues from mastcell-deficient (Ws/Ws) and their littermate (+/+) control rats (right panels) were exposed to corticotropin-releasing factor (10�6 M,black bars), sauvagine (10�9 M, gray bars) or saline (empty bars). The increase in short-circuit current (Isc) above baseline and the fluxof horseradish peroxidase (HRP) were measured. Values indicate the mean � S.E.M., n = 8—12 tissues/group. *P < 0.05 vs. saline.

Table 2 Effect of CRF and SVG on colonic RMCP II release

Basal Treatment

Saline CRF (10�6 M) SVG (10�9 M)

RMCP II (ng/g tissue) 167.5 � 23.1 521.7 � 79.3 2364.3 � 397.1 * 2623.9 � 465.4 *

Four adjacent segments (exposed area, 0.6 cm � 0.6 cm) from the distal colon of WKY rats were mounted in Ussing chambers. Total RMCP IIconcentration in mixed samples from serosal (0.25 ml) + mucosal (0.25 ml) buffers was measured in one segment at equilibrium (basalrelease), and 1 h after the serosal addition of either corticotropin-releasing factor (CRF), sauvagine (SVG), or corresponding vehicle (saline)in the remaining segments. Values indicate the mean � S.E.M., n = 8 tissues/treatment; *P < 0.01 vs. saline.

Stress-mast cell intraepithelial networks regulate colonic barrier 1253

and to SVG at 10�9 M (18.9 � 3.9 pmol h�1 cm�2;4.4 � 2.2 pmol h�1 cm�2, P < 0.05, vehicle vs. doxantrazole).Furthermore, CRF at 10�6 M and SVG at 10�9 M increased by4.5-fold and 5.0-fold, respectively, the release of RMCP II intotissue buffers, as compared to vehicle (Table 2), indicating theparticipation of mucosal mast cells.

When CRF 10�6 M or SVG 10�9 M were added to tissuesfromWs/Ws rats, the Isc increase was significantly reduced by40% and 60%, respectively, compared with responses in tis-sues from +/+ rats (Fig. 5). Notably, no increase in the flux ofHRP was observed in tissues from Ws/Ws rats in response toCRF and SVG, whereas amarked enhancement appeared in +/+ tissues (Fig. 5). Collectively, these findings support theinvolvement of mast cells in epithelial responses to stress-related peptides.

4. Discussion

In this study, we have extended our previous findings (Santoset al., 1999; Saunders et al., 2002; Wallon et al., 2008) toshow that the stress-like peptides CRF and SVG stimulated ionsecretion, as indicated by the increase in Isc, and macro-molecular permeability, as indicated by the enhanced flux ofHRP, in the distal colon of WKY rats in vitro. Our resultsindicate the involvement of mucosal/submucosal CRF recep-tors, as determined by the significant reduction of bothresponses in tissues pretreated with astressin, a non-selec-

tive CRF receptor antagonist. In addition, we found that theeffects on epithelial function are also mast cell-dependent,as shown by their blockade by doxantrazole in WKY, andinhibition in Ws/Ws rats. Furthermore, both CRF and SVGenhanced the release of RMCP II suggesting the participationof mucosal mast cells.

CRF and SVG share a 40—50% structural homology andwhile both exhibit equal affinity for CRF1, SVG displays 10—40-fold higher affinity for CRF2 (�1 nM) than CRF (12—190 nM) (Grigoriadis et al., 1996; Hoare et al., 2005). Wefound that, on a molar basis, maximal epithelial responses toSVG were achieved with doses 200—1000-fold lower thanCRF, suggesting CRF2-predominant effect. This is in line withstudies reporting that SVG was more potent (20—1000-fold)than CRF to dilate mesenteric arterial beds in the rat(Lederis et al., 1985) or to stimulate [Ca2+] increase inhuman epidermoid A-431 cells (Kiang, 1997). Interestingly,the response to SVG was accompanied by a dose-dependentbell-shaped effect where the excitatory action appeared at10�10 M, peaked at 10�9 M, and decreased thereafter. Weignore the precise mechanism involved but this phenomenonhas been often described for other G protein-coupled recep-tors (Pao and Benovic, 2002). Although it could be due toaltered expression or receptor desensitization, in response toconformational changes or variation of intracellular cAMP orCa2+ levels, induced by non-specific or counter-regulatoryauto/paracrine co-release of interfering molecules, other

1254 J. Santos et al.

mechanisms have been also implicated (Mancinelliet al.,1998; Kanno et al., 1999).

The effect of both peptides on colonic Isc and HRP flux wasinhibited by astressin. Astressin is equally potent at CRF1 andCRF2 (Gulyas et al., 1995) exhibiting similar to greaterpotency than any other CRF peptide antagonist (Hillhouseand Grammatopoulos, 2006). We found that the inhibitoryeffect of astressin was fully displayed when used in 10-foldexcess concentration of CRF and SVG. This observation, alongwith the previously reported 5-fold higher antagonist-to-agonist ratio for astressin-to-SVG than for astressin-to-CRF,to block gastric emptying (Martinez et al., 1998), reinforcesthe possibility of a predominant CRF2-mediated effect.Indeed, and consistent with our results, a prosecretory andproinflammatory effect of CRF2 has been described in theileum of CRF2-null mice, in response to C. difficile toxin A(Kokkotou et al., 2006).

We did not look at pathways involved in HRP passage.However, previous reports have shown that the enhancementof rat colonic epithelial permeability after chronic stress orsystemic administration of CRH involves complex neuro-immune interactions that activate both paracellular andtranscellular pathways (Santos et al., 1999, 2001). Althoughstill unclear, recent findings suggest that while CRF1 regulatesthe paracellular route (Barreau et al., 2007) CRF2 regulatesthe transcellular passage (Gareau et al., 2007).

Our present observations demonstrate that doxantrazoleprevented the enhancement of both Isc and HRP permeabilityto CRF-like peptides in vitro. Furthermore, colonic segmentsfrom Ws/Ws exhibited reduced responses to these peptides,compared with tissues from the mast cell-replete +/+ rats.We showed previously that i.p. CRF-induced colonic epithe-lial responses in WKY rats were also mast cell-dependent(Santos et al., 1999), a finding recently replicated (Barreauet al., 2007). Colonic barrier unresponsiveness to stress hasbeen previously reported in mast cell-deficient rodents (Cas-tagliuolo et al., 1998; Santos et al., 2001) as well as thereversal of epithelial responsiveness to stress after mast cellreconstitution (Castagliuolo et al., 1998), highlighting therole of mast cells in stress-related epithelial physiology.Moreover, a similar role has been recently confirmed inhuman colonic biopsies, in which CRF-enhanced HRP perme-ability was abolished by lodoxamide in vitro (Wallon et al.,2008). It could be argued that other phenotypic abnormal-ities of Ws/Ws, such as the deficiency of melanocytes, ery-throcytes and interstitial cells of Cajal (Niwa et al., 1991),could be responsible, at least in part, of the reducedresponses to CRF and SVG. However, a major contributionseems unlikely because our in vitro preparation excludes thepresence of melanocytes and interstitial cells of Cajal, and,ten weeks after birth, when we performed the experiments,Ws/Ws have recovered the number of erythrocytes (Niwaet al., 1991).

In this study, we did perform partial stripping, a procedurecommonly associated with the retainment of muscularismucosae and submucosa (Andres et al., 1985), where differ-ent mast cell subtypes reside. Mucosal mast cells contain andgenerate mediators, such as RMCP II, histamine, 5-hydroxy-tryptamine, proteases, lipid mediators, and nerve-growthfactor that influence intestinal epithelial secretion and per-meability (Santos and Perdue, 2001; Barreau et al., 2007). Wefound increased release of RMCP II in response to both CRF

and SVG indicating the activation of mucosal mast cells.Previous reports, using immunohistochemical and ultrastruc-tural imaging techniques, and selective pharmacologicalblockade, have also shown the participation of mucosal mastcells on stress-induced barrier abnormalities (Santos et al.,2001; Soderholm et al., 2002b; Barreau et al., 2007, 2008).Although these studies support a prominent role for mucosalmast cells, they do not exclude, as ours, the participation ofsubmucosal mast cells.

Our findings indicate that colonic epithelial responses maybe more sensitive to SVG than CRF indicating either a CRF2-predominant effect or the presence of still non-character-ized subclasses of the CRF receptor with higher affinity forSVG. Both CRF1 and CRF2 mRNA are prominently expressed inthe human and rat colon (Kostich et al., 1998; Chatzaki et al.,2004). However, WKY rats over-express CRF2 in some periph-eral organs (Makino et al., 1998) and some studies point atCRF2 as the main functional receptor in these organs (Loven-berg et al., 1995). In fact, CRF2(b) is widely expressed in thegastrointestinal tract in rodents, and is potently activated bySVG, and about 10-fold stronger in second messenger gen-eration than CRF2(a) or CRF2(c) (Hillhouse and Grammatopou-los, 2006) while CRF2(a) was mainly localized in the luminalsurface of the crypts in the colon of naıve Sprague—Dawleyrats (Lovenberg et al., 1995; Chatzaki et al., 2004). Inhumans, CRF2 mRNA and protein are expressed in culturedcolonic epithelial cells and in colonocytes in patients withulcerative colitis, and up-regulated upon proinflammatorystimuli (Kawahito et al., 1995; Moss et al., 2007), and CRF2(a)is the predominant isoform in HT-29 cells (Kokkotou et al.,2006). Since, mast cells can be activated by a vast array ofmolecules released by immune and epithelial cells, ourresults could be explained, at least in part, by secondarymast cell activation by alternative molecules released by CRFreceptor-bearing neighboring cells.

Although CRF receptors are expressed in resident mastcells in certain locations such as the skin (Donelan et al.,2006), its presence in rat colonicmast cells is still unresolved,although CRF1 immuno-staining has been detected in somenon-specified lamina propria cells in the rat colon (Lovenberget al., 1995; Chatzaki et al., 2004). In contrast, CRF1 and CRF2have been recently described on subepithelial mast cells inhuman colonic biopsies from healthy individuals (Wallonet al., 2008) and both, the human leukemic mast cell lineand human umbilical cord blood-derived mast cells, displayCRF1 and CRF2, activation of which leads to the selectiverelease of mediators (Cao et al., 2005). Human mast cellssynthesize and secrete CRF and urocortin in response to IgE(Kempuraj et al., 2004) and both CRF1 and CRF2 are up-regulated in these cells, in inflammatory disorders (McEvoyet al., 2001; Papadopoulou et al., 2005). Moreover, CRF-mediated activation of mast cells has been shown to regulateepithelial permeability in the human colon and skin (Cromp-ton et al., 2003; Wallon et al., 2008). Finally, our tissues wereobtained after gentle surgical manipulation, a process thatresults in a progressive activation and increase of residentmast cells in the rat and human intestine (Kalff et al., 1999;The et al., 2008). Therefore, based on above observations,our results suggest the involvement of a direct effect of CRFand SVG on mast cells in epithelial response.

In conclusion, we have shown that stress-likepeptides stimulated epithelial ion secretion and enhanced

Stress-mast cell intraepithelial networks regulate colonic barrier 1255

macromolecular permeability in the rat colon. This responsewas mediated by both CRF receptors and mast cells andhighlight the importance of local regulatory circuits in thecontrol of stress-promoted epithelial pathophysiology. Sincestress and mast cells seem to be involved in both irritablebowel syndrome and inflammatory bowel disease, betterunderstanding of mucosal regulatory networks, using newlydeveloped and more specific agonists/antagonists for CRFreceptor subtypes along with clear definition of the functionand anatomical distribution of CRF receptors and variantsmay provide newer targets for the management of thesedisorders.

Role of the funding sources

The Spanish Ministry of Sanidad y Consumo, SubdireccionGeneral de Investigacion Sanitaria, Instituto Carlos III, Fondode Investigacion Sanitaria & the Crohn’s and Colitis Founda-tion of Canada provide uninterested funding support fordevelopment of biomedical research with no further rolein study design; in the collection, analysis and interpretationof data; in the writing of this report; and in the decision tosubmit the paper for publication to this journal.

Conflict of interest

The corresponding author, on behalf of all authors, declareshaving no competing interests.

Acknowledgements

Supported in part by the Spanish Ministry of Sanidad y Con-sumo, Subdireccion General de Investigacion Sanitaria, Insti-tuto Carlos III, Fondo de Investigacion Sanitaria (PI05/1423,Santos, J.; CD05/00060, Vicario, M.; BF03/00392, Alonso, C.)& the Crohn’s and Colitis Foundation of Canada (Perdue,M.H.).

Contributors: The Corresponding Author declares that allauthors contributed in a substantial way to the present studyand have approved the final manuscript.

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