Psychoneuroendocrinology (2008) 33, 1248—1256
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n
Stress neuropeptides evoke epithelial responses via 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 a
Digestive Diseases Research Unit, Institut de Recerc¸a Vall d’Hebron, Department of Gastroenterology, Hospital Universitari Vall d’Hebron, Universitat Auto `noma de Barcelona, Department of Medicine, Barcelona, Spain b Intestinal Disease Research Programme, Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada Received 9 January 2008; received in revised form 11 June 2008; accepted 1 July 2008
KEYWORDS Corticotropin-releasing factor; 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 permeability in the rat colon, and mast cells were involved. However, the ability of CRF-sensitive mucosal/submucosal loops to regulate intestinal barrier and the participation of resident mast cells 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 segments mounted 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 in response to CRF [10 6 to 10 8 M] or sauvagine [10 8 to 10 10 M] in tissues pretreated with astressin, doxantrazole, or vehicle. Results: Stress-like peptides (sauvagine > CRF) induced a dose-dependent increase in shortcircuit current (maximal at 30 min), and significantly enhanced horseradish peroxidase flux and protease II release in WKY. Epithelial responses were inhibited by both astressin and doxantrazole, and significantly reduced in tissues from Ws/Ws rats. Conclusion: The stress mediators CRF and sauvagine modulate barrier function in the rat colon acting on mucosal/submucosal CRF receptor-bearing cells, through mast cell-dependent pathways. # 2008 Elsevier Ltd. All rights reserved.
Abbreviations: CRF, corticotropin-releasing factor; HRP, horseradish peroxidase; Isc, short-circuit current; NS, not significant; RMCP II, rat mast cell protease II; SVG, sauvagine; WKY, Wistar—Kyoto. * Corresponding author at: Digestive Diseases Research Unit, Institut de Recerca Vall d’Hebron, Department of Gastroenterology, Hospital Universitari Vall d’Hebron, Universitat Auto `noma de Barcelona, Barcelona, Spain. Fax: +34 934894032. E-mail address:
[email protected] (J. Santos).
1. Introduction A number of studies in humans and experimental animals now indicate that stress is associated with abnormalities in visceral 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
0306-4530/$ — see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2008.07.002
Stress-mast cell intraepithelial networks regulate colonic barrier and large bowel. These findings may help to explain the proposed role of stress in the modulation of mucosal inflammation and its putative participation in physiopathological events underlying common gastrointestinal disorders such as irritable bowel syndrome (Bennett et al., 1998) and inflammatory bowel disease (Mawdsley and Rampton, 2005). However, the pathways and molecular mechanisms by which stress induces intestinal dysfunction remain unclear. The classical view of the intestinal epithelium as a mere physical barrier between the luminal content and the internal milieu also involved in digestion and absorption of nutrients has been overtaken by its increasingly recognized role as an active player in the modulation of local inflammatory events and gut functional homeostasis (Fiocchi, 1997; Sansonetti, 2004). Growing and compelling evidence shows that stress regulates epithelial function. Corticotropin-releasing factor (CRF), a 41-amino acid peptide, and related analogues like the amphibian 40-amino acid peptide sauvagine (SVG), mediate endocrine/immune, autonomic, visceral and behavioral responses to stress acting on high affinity membranebound receptors on target cells (Bale and Vale, 2004). Two main receptor subtypes, CRF1 and CRF2, have been characterized in mammals (Turnbull and Rivier, 1997) but while CRF and SVG have similar affinity for CRF1, SVG displays higher affinity for CRF2 than CRF (Grigoriadis et al., 1996). Others and we have shown that stress stimulates epithelial ion secretion and permeability (Santos et al., 1998; Barreau et al., 2004; Alonso et al., 2008) and modulates mucosal immune and inflammatory responses (So ¨derholm et al., 2002a; Yang et al., 2006) in human and rat small intestine and colon. Moreover, peripheral CRF provoked transport and barrier abnormalities resembling those observed in the intestine of stressed rats (Santos et al., 1999), effects that were inhibited by the non-selective CRF antagonist, a-helical CRF9-41 (Saunders et al., 2002; So ¨derholm et al., 2002b). Mast cells regulate barrier physiology in normal as well as inflamed intestine, in rats and humans (Crowe et al., 1997; Berin et al., 1998). Furthermore, they play a key role in stressmediated intestinal epithelial responses, including ion secretion, transport of macromolecules and mucin release, possibly via CRF-activated pathways (Castagliuolo et al., 1996; Barreau et al., 2008). Despite convincing evidence of intestinal expression of CRF-like peptides and receptors and its production by local immunocytes and enteroendocrine cells (Chatzaki et al., 2004; Tache and Bonaz, 2007), only recently a role for subepithelial CRF-mast cell loops in the regulation of colonic permeability in human biopsies has been reported (Wallon et al., 2008). However, the contribution of mucosal/submucosal CRF-based autocrine/paracrine networks and the involvement of resident mast cells in the control of barrier function in the rat intestine are still unresolved. The aims of the present study were to examine the ability of CRF-sensitive mucosal/submucosal circuits to regulate epithelial physiology in the rat colon, and to determine the participation of resident mast cells in these responses.
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Constant, QC, Canada). Some studies involved the use of mast cell-deficient Ws/Ws rats and their normal +/+ littermate 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/Ws rats have a 12-base deletion in the tyrosine kinase domain of the c-kit gene (Tsujimura et al., 1991) that results in the absence of mast cells and melanocytes and a reduced number of erythrocytes. By ten weeks of age erythrocyte numbers are greatly recovered in Ws/Ws rats (Niwa et al., 1991), which also 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 mast cells (Berin et al., 1998). Rats, housed two per cage, were maintained on a normal 12:12-h dark/light cycle and provided with food and water ad libitum. The Animal Care Committee at McMaster University approved all procedures.
2.2. Epithelial measurements in Ussing chambers
2. Methods
After two weeks of daily handling by the same investigator (to avoid inadvertent stress from human contact), rats were euthanized by decapitation. The distal colon was removed, placed in 37 8C oxygenated Krebs, stripped of longitudinal muscle and myenteric plexus and opened along the mesenteric border. Four adjacent pieces from each rat were mounted in Ussing chambers (World Precision Instruments, Sarasota, FL). The chamber opening exposed 0.6 cm2 of tissue surface area to 8 ml of circulating oxygenated Krebs buffer 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 an energy source, osmotically balanced by 10 mM mannitol in the mucosal buffer. The chambers contained agar-salt bridges to monitor the potential difference across the tissue and to inject the required short-circuit current (Isc) to maintain a zero potential difference as registered via an automated voltage clamp (World Precision Instruments). Isc (in mA/cm2), a measure of net active ion transport, was recorded by a computer connected to the voltage-clamp system. Tissue conductance, an indicator of ion permeability and tissue viability, was calculated according to Ohm’s law and expressed as milli-Siemens/cm2. Uptake of macromolecules was assessed by measuring the mucosal-to-serosal flux of horseradish peroxidase (HRP). HRP (type VI, Sigma Chemical Co., St. Louis, MO) was added at 10 5 M to the luminal buffer 20 min after the tissues were mounted, and allowed to equilibrate for 30 min. Serosal samples (0.5 ml) were then obtained at 30-min intervals for 2 h (volume in the chamber maintained by replacing with glucose buffer). HRP activity was determined by a modified Worthington method as previously described (Santos et al., 1999). The mucosal-to-serosal flux of HRP was calculated according to a standard formula (Saunders et al., 2002) and expressed as picomoles per hour per square centimeter.
2.1. Animals
2.3. Drugs and treatments
Experiments were performed on tissues from male Wistar— Kyoto (WKY) rats (200—250 g; Charles River Laboratories, St.
Rat/human CRF (Peninsula Laboratories, Inc., Belmont, CA), ‘‘skin frog’’ SVG, astressin, cyclo-(30-33)-[D-Phe12,Nle12,N-
1250 le21,38,Glu30,Lys33] r/h CRF-(12-41) (kindly donated by Dr. J. Rivier, Salk Institute, Clayton Foundation Laboratories for Peptide Biology, La Jolla, CA), and doxantrazole (a gift from Burroughs Wellcome Co., Research Triangle Park, NC) were dissolved following manufacturers’ instructions, aliquoted and kept frozen at 80 8C until used. Astressin displays high affinity for both CRF receptor subtypes and is more potent than a-helical CRF12-41 in blocking various CRF- and SVG-mediated effects (Gulyas et al., 1995; Hillhouse and Grammatopoulos, 2006). Rat mast cell protease II (RMCP II) levels were also evaluated in some experiments to clarify the participation of mucosal mast cells. RMCP II was measured by ELISA (Moredun Sientific Ltd., Midlothian, Scotland) in 0.5 ml buffer samples (0.25 ml serosal + 0.25 ml mucosal), as described (Vergara et al., 2002). RMPC II concentration 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 used in some studies.
J. Santos et al. parations (Kiang, 1997; Santos et al., 1999). RMCP II was measured in separate experiments at equilibrium and 1h after CRF [10 6 M], SVG [10 9 M] or saline. To ascertain the role of mast cells, tissues from Ws/Ws and +/+ rats were treated with the maximal effective doses of CRF and SVG. Treatment assignment for astressin, doxantrazole and Ws/Ws and +/+ experiments was performed in a way similar as that described before.
2.5. Statistical analysis Results are expressed as means S.E.M. unless otherwise stated. For each tissue, Isc and conductance responses were calculated by subtracting baseline values from maximum values after treatment, and expressed as the increment (D) for that period. For each treatment, paired statistical comparisons were performed. Multiple groups were compared by Dunnett’s test and Tukey—Kramer test following a significant one-way analysis of variance. Single comparisons were performed by paired or unpaired Student’s t-test where appropriate. P < 0.05 was considered significant.
2.4. Experimental design
3. Results Baseline values for Isc and conductance were calculated at equilibrium, 20 min after the tissues were mounted, and then every 30 min for 2 h. The HRP flux was determined over at least two stable 30-min periods after equilibration. Tissues from each rat were matched according to baseline Isc and conductance (paired tissues not differing by more than 20%). Then, tissue pairs were exposed to the active drug (CRF/SVG) or vehicle (sterile saline for both). Treatment assignment was based on a triple restricted randomization process: (A) One tissue pair from each rat was randomly allocated to receive either CRF [10 6 to 10 8 M]/ vehicle (1) or SVG [10 8 to 10 10 M]/vehicle (2), using a computerized random number generator tool with no repeats (1 or 2); the second tissue pair was then allocated, by minimization, to the remaining option; (B) the first tissue of each pair was randomly allocated to receive the active drug (1) or its vehicle (2), whereas the second tissue 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 tissue pairs were exposed to just different doses of active drugs selected as described in (B), until groups (n = 12—16) were completed. Drugs and vehicles were administered on the serosal side of the tissues. In preliminary experiments, an enzyme inhibitor cocktail, known to prevent peptide digestion in similar systems, was added to the buffer:bacitracin [2 10 5 M], leupeptin [9 10 6 M], phosphoramidon [2 10 6 M], and aprotinin [500 kallikrein-inactivating units/ml]. Because electrophysiological responses to the peptides were not affected by this treatment, we did not use protease inhibitors in subsequent experiments. Additional tissues were exposed to astressin [10 5 to 10 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 doxantrazole were based on previous reports showing their ability to induce or inhibit a variety of electrophysiological, biochemical and morphological responses in different in vitro pre-
3.1. Effect of CRF and SVG on colonic shortcircuit current All tissue groups used to evaluate drug and vehicle responses displayed equal Isc and conductance baseline values (Table 1). The typical Isc response CRF and SVG showed a dose-dependent gradual increase detectable within 3 min, which reached a maximum at 30 min, and decreased but remained above baseline thereafter (Fig. 1). The increase in Isc was paralleled by an increase in potential difference so that conductance did not change significantly at any point. The response to CRF ranged from the peak average increment above 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% for vehicle (Fig. 2A). The predominant CRF2 agonist SVG (Fig. 2B) also increased Isc above baseline by 64% at 10 8 M, by 114% at 10 9 M, and by 48% at 10 10 M, as compared to 12.5% for vehicle (P < 0.05 for all comparisons).
3.2. Effect of CRF and SVG on HRP flux across the colonic mucosa HRP flux was enhanced by CRF in a concentration-dependent manner (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-significant increase at lower CRF concentrations, compared to tissues treated with vehicle (3.9 0.9 pmol h 1 cm 2). SVG also induced a marked increase in HRP flux that peaked 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 concentrations of SVG were not associated with further enhancement in HRP flux (Fig. 3B).
3.3. Effect of the CRF antagonist astressin on CRF-and SVG-stimulated colonic Isc and HRP flux Astressin, when added at different concentrations to the serosal compartment, did not alter either Isc or HRP flux,
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Stress-mast cell intraepithelial networks regulate colonic barrier Table 1 Rats
Baseline electrophysiological characteristics of tissues used in experiments Drug
Isc (mA/cm2)
G (mS/cm2)
n
Value
Range
P
Value
Range
P
WKY
CRF SVG Vehicle Astressin Vehicle DOX Vehicle
39.9(14.5) 37.5(15.1) 37.3(15.0) 36.7(14.4) 36.4(13.1) 38.8(13.1) 37.4(13.5)
12.8—64.3 10.2—64.4 12.0—63.0 11.2—64.2 13.5—61.4 16.3—64.3 16.6—63.3
0.89
21.2(6.1) 22.7(6.2) 20.9(6.8) 22.4(6.9) 23.3(6.8) 22.5(5.3) 21.5(6.8)
12.0—35.2 10.6—37.8 11.5—36.6 11.5—38.6 12.3—36.5 16.5—36.8 12.3—37.1
0.54
68 68 40 68 32 28 28
Ws/Ws
CRF SVG Vehicle
34.8(9.4) 35.6(9.5) 35.3(8.7)
20.3—52.5 19.3—48.8 18.5—48.6
0.98
25.7(7.1) 25.9(7.5) 24.5(4.0)
16.7—38.5 15.7—38.8 18.5—31.5
0.85
8 8 12
+/+
CRF SVG Vehicle
35.7(10.6) 35.4(11.8) 36.2(8.7)
20.9—51.2 18.8—52.1 19.8—50.6
0.98
26.3(4.4) 24.5(5.8) 24.9(3.2)
21.3—34.8 17.0—33.2 21.0—31.6
0.70
8 8 12
Data values are expressed as mean (S.D.). No statistical differences were observed for Isc or G comparisons within rat species and treatment subgroups, 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.
compared with its vehicle. Astressin, when administered in 10fold 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 HRP flux increase (14.5 2.9 pmol h 1 cm 2; 5.7 1.3 pmol h 1 cm 2, P < 0.05, vehicle vs. astressin) in response to CRF 10 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) and HRP flux increase (21.9 3.8 pmol h 1 cm 2; 10.4 1.6 pmol h 1 cm 2, P < 0.05, vehicle vs. astressin) in response to SVG 10 9 M (Fig. 4). These results indicate that epithelial responses to CRF and SVG involve specific CRF receptor activation.
3.4. Role of intestinal mast cells in epithelial responses to CRF and SVG The mast cell stabilizer doxantrazole at 10 5 M did not change Isc or HRP flux compared to its vehicle. However, doxantrazole abolished 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). Similarly, 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),
Figure 1 Effect of corticotropin-releasing factor (CRF) and sauvagine (SVG) on colonic mucosal electrophysiology. Tracings show representative changes in short-circuit current (Isc) and conductance (vertical pulses on top of tracings) in segments from the distal colon of WKY rats mounted in Ussing chambers after the serosal exposure to most effective doses of CRF, SVG and vehicle (saline). Both peptides induced a marked and gradual increase in Isc, which peaked after 30 min and slowly decreased thereafter but remained elevated above baseline. Conductance was not affected.
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Figure 2 Dose—response curves for corticotropin-releasing factor (CRF) and sauvagine (SVG) on colonic short-circuit current (Isc). Distal colonic tissues from WKY rats were mounted in Ussing chambers. Different concentrations of CRF (upper panel, A), SVG, (lower panel, B), or vehicle were administered on the serosal side at equilibrium (baseline) and Isc recorded at baseline and 30 min later. Values are expressed as the averaged mean increments (D) above baseline S.E.M. for each CRF/SVG concentration, n = 12—16 tissues/group. *P < 0.05 vs. Dvehicle (D for CRF vehicle: 6.62 2.4 mA/cm2; D for SVG vehicle: 4.4 2.2 mA/cm2, n = 20 tissues/group).
J. Santos et al.
Figure 3 Dose—response curves for corticotropin-releasing factor (CRF) and sauvagine (SVG) on horseradish peroxidase (HRP) flux across the colonic mucosa. Distal colonic tissues from WKY rats were mounted in Ussing chambers. Different concentrations 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 2 consecutive 30-min stable flux periods. Values indicate the mean S.E.M. for each CRF/SVG concentration, n = 8—16 tissues/group. *P < 0.05 vs. vehicle (CRF vehicle: 3.9 0.9 pmol 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 colonic tissues from WKY rats mounted in Ussing chambers were exposed to increasing concentrations of astressin administered on the serosal side, 30 min before the addition of 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 flux of horseradish peroxidase (HRP) were measured. Values indicate the mean S.E.M., n = 8—16 tissues/group; *P < 0.05 vs. vehicle.
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Stress-mast cell intraepithelial networks regulate colonic barrier
Figure 5 Role of intestinal mast cells on corticotropin-releasing factor- and sauvagine-induced colonic epithelial responses. Distal segments from WKY rats (left panels), pretreated ( 30 min) with serosal doxantrazole/vehicle, as well as untreated tissues from mast cell-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 flux of 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
RMCP II (ng/g tissue)
167.5 23.1
Treatment 6
M)
Saline
CRF (10
521.7 79.3
2364.3 397.1 *
SVG (10
9
M)
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 II concentration in mixed samples from serosal (0.25 ml) + mucosal (0.25 ml) buffers was measured in one segment at equilibrium (basal release), 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.
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 by 4.5-fold and 5.0-fold, respectively, the release of RMCP II into tissue buffers, as compared to vehicle (Table 2), indicating the participation of mucosal mast cells. When CRF 10 6 M or SVG 10 9 M were added to tissues from Ws/Ws rats, the Isc increase was significantly reduced by 40% and 60%, respectively, compared with responses in tissues from +/+ rats (Fig. 5). Notably, no increase in the flux of HRP was observed in tissues from Ws/Ws rats in response to CRF and SVG, whereas a marked enhancement appeared in +/ + tissues (Fig. 5). Collectively, these findings support the involvement of mast cells in epithelial responses to stressrelated peptides.
4. Discussion In this study, we have extended our previous findings (Santos et al., 1999; Saunders et al., 2002; Wallon et al., 2008) to show that the stress-like peptides CRF and SVG stimulated ion secretion, as indicated by the increase in Isc, and macromolecular permeability, as indicated by the enhanced flux of HRP, in the distal colon of WKY rats in vitro. Our results indicate the involvement of mucosal/submucosal CRF receptors, as determined by the significant reduction of both responses in tissues pretreated with astressin, a non-selec-
tive CRF receptor antagonist. In addition, we found that the effects on epithelial function are also mast cell-dependent, as shown by their blockade by doxantrazole in WKY, and inhibition in Ws/Ws rats. Furthermore, both CRF and SVG enhanced the release of RMCP II suggesting the participation of mucosal mast cells. CRF and SVG share a 40—50% structural homology and while 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). We found that, on a molar basis, maximal epithelial responses to SVG were achieved with doses 200—1000-fold lower than CRF, suggesting CRF2-predominant effect. This is in line with studies 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 in human epidermoid A-431 cells (Kiang, 1997). Interestingly, the response to SVG was accompanied by a dose-dependent bell-shaped effect where the excitatory action appeared at 10 10 M, peaked at 10 9 M, and decreased thereafter. We ignore the precise mechanism involved but this phenomenon has been often described for other G protein-coupled receptors (Pao and Benovic, 2002). Although it could be due to altered expression or receptor desensitization, in response to conformational changes or variation of intracellular cAMP or Ca2+ levels, induced by non-specific or counter-regulatory auto/paracrine co-release of interfering molecules, other
1254 mechanisms have been also implicated (Mancinelli et al.,1998; Kanno et al., 1999). The effect of both peptides on colonic Isc and HRP flux was inhibited by astressin. Astressin is equally potent at CRF1 and CRF2 (Gulyas et al., 1995) exhibiting similar to greater potency than any other CRF peptide antagonist (Hillhouse and Grammatopoulos, 2006). We found that the inhibitory effect of astressin was fully displayed when used in 10-fold excess concentration of CRF and SVG. This observation, along with the previously reported 5-fold higher antagonist-toagonist ratio for astressin-to-SVG than for astressin-to-CRF, to block gastric emptying (Martinez et al., 1998), reinforces the possibility of a predominant CRF2-mediated effect. Indeed, and consistent with our results, a prosecretory and proinflammatory effect of CRF2 has been described in the ileum 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 enhancement of rat colonic epithelial permeability after chronic stress or systemic administration of CRH involves complex neuroimmune interactions that activate both paracellular and transcellular pathways (Santos et al., 1999, 2001). Although still unclear, recent findings suggest that while CRF1 regulates the paracellular route (Barreau et al., 2007) CRF2 regulates the transcellular passage (Gareau et al., 2007). Our present observations demonstrate that doxantrazole prevented the enhancement of both Isc and HRP permeability to CRF-like peptides in vitro. Furthermore, colonic segments from 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 epithelial responses in WKY rats were also mast cell-dependent (Santos et al., 1999), a finding recently replicated (Barreau et al., 2007). Colonic barrier unresponsiveness to stress has been previously reported in mast cell-deficient rodents (Castagliuolo et al., 1998; Santos et al., 2001) as well as the reversal of epithelial responsiveness to stress after mast cell reconstitution (Castagliuolo et al., 1998), highlighting the role of mast cells in stress-related epithelial physiology. Moreover, a similar role has been recently confirmed in human colonic biopsies, in which CRF-enhanced HRP permeability was abolished by lodoxamide in vitro (Wallon et al., 2008). It could be argued that other phenotypic abnormalities of Ws/Ws, such as the deficiency of melanocytes, erythrocytes and interstitial cells of Cajal (Niwa et al., 1991), could be responsible, at least in part, of the reduced responses to CRF and SVG. However, a major contribution seems unlikely because our in vitro preparation excludes the presence 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 (Niwa et al., 1991). In this study, we did perform partial stripping, a procedure commonly associated with the retainment of muscularis mucosae and submucosa (Andres et al., 1985), where different mast cell subtypes reside. Mucosal mast cells contain and generate mediators, such as RMCP II, histamine, 5-hydroxytryptamine, proteases, lipid mediators, and nerve-growth factor that influence intestinal epithelial secretion and permeability (Santos and Perdue, 2001; Barreau et al., 2007). We found increased release of RMCP II in response to both CRF
J. Santos et al. and SVG indicating the activation of mucosal mast cells. Previous reports, using immunohistochemical and ultrastructural imaging techniques, and selective pharmacological blockade, have also shown the participation of mucosal mast cells on stress-induced barrier abnormalities (Santos et al., 2001; So ¨derholm et al., 2002b; Barreau et al., 2007, 2008). Although these studies support a prominent role for mucosal mast cells, they do not exclude, as ours, the participation of submucosal mast cells. Our findings indicate that colonic epithelial responses may be more sensitive to SVG than CRF indicating either a CRF2predominant effect or the presence of still non-characterized subclasses of the CRF receptor with higher affinity for SVG. Both CRF1 and CRF2 mRNA are prominently expressed in the human and rat colon (Kostich et al., 1998; Chatzaki et al., 2004). However, WKY rats over-express CRF2 in some peripheral organs (Makino et al., 1998) and some studies point at CRF2 as the main functional receptor in these organs (Lovenberg et al., 1995). In fact, CRF2(b) is widely expressed in the gastrointestinal tract in rodents, and is potently activated by SVG, and about 10-fold stronger in second messenger generation than CRF2(a) or CRF2(c) (Hillhouse and Grammatopoulos, 2006) while CRF2(a) was mainly localized in the luminal surface of the crypts in the colon of naı¨ve Sprague—Dawley rats (Lovenberg et al., 1995; Chatzaki et al., 2004). In humans, CRF2 mRNA and protein are expressed in cultured colonic epithelial cells and in colonocytes in patients with ulcerative colitis, and up-regulated upon proinflammatory stimuli (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 of molecules released by immune and epithelial cells, our results could be explained, at least in part, by secondary mast cell activation by alternative molecules released by CRF receptor-bearing neighboring cells. Although CRF receptors are expressed in resident mast cells in certain locations such as the skin (Donelan et al., 2006), its presence in rat colonic mast cells is still unresolved, although CRF1 immuno-staining has been detected in some non-specified lamina propria cells in the rat colon (Lovenberg et al., 1995; Chatzaki et al., 2004). In contrast, CRF1 and CRF2 have been recently described on subepithelial mast cells in human colonic biopsies from healthy individuals (Wallon et al., 2008) and both, the human leukemic mast cell line and human umbilical cord blood-derived mast cells, display CRF1 and CRF2, activation of which leads to the selective release of mediators (Cao et al., 2005). Human mast cells synthesize and secrete CRF and urocortin in response to IgE (Kempuraj et al., 2004) and both CRF1 and CRF2 are upregulated in these cells, in inflammatory disorders (McEvoy et al., 2001; Papadopoulou et al., 2005). Moreover, CRFmediated activation of mast cells has been shown to regulate epithelial permeability in the human colon and skin (Crompton et al., 2003; Wallon et al., 2008). Finally, our tissues were obtained after gentle surgical manipulation, a process that results in a progressive activation and increase of resident mast 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 CRF and SVG on mast cells in epithelial response. In conclusion, we have shown that stress-like peptides stimulated epithelial ion secretion and enhanced
Stress-mast cell intraepithelial networks regulate colonic barrier macromolecular permeability in the rat colon. This response was mediated by both CRF receptors and mast cells and highlight the importance of local regulatory circuits in the control of stress-promoted epithelial pathophysiology. Since stress and mast cells seem to be involved in both irritable bowel syndrome and inflammatory bowel disease, better understanding of mucosal regulatory networks, using newly developed and more specific agonists/antagonists for CRF receptor subtypes along with clear definition of the function and anatomical distribution of CRF receptors and variants may provide newer targets for the management of these disorders.
Role of the funding sources The Spanish Ministry of Sanidad y Consumo, Subdireccio ´n General de Investigacio ´n Sanitaria, Instituto Carlos III, Fondo de Investigacio ´n Sanitaria & the Crohn’s and Colitis Foundation of Canada provide uninterested funding support for development of biomedical research with no further role in study design; in the collection, analysis and interpretation of data; in the writing of this report; and in the decision to submit the paper for publication to this journal.
Conflict of interest The corresponding author, on behalf of all authors, declares having no competing interests.
Acknowledgements Supported in part by the Spanish Ministry of Sanidad y Consumo, Subdireccio ´n General de Investigacio ´n Sanitaria, Instituto Carlos III, Fondo de Investigacio ´n 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 all authors contributed in a substantial way to the present study and have approved the final manuscript.
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