1 Intestinal Disease Research Program, Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and 2 Institute of Neurobiology, University of Amsterdam, 1098SM Amsterdam, The Netherlands
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ABSTRACT |
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We examined the
effect of stress on colonic epithelial physiology, the role of
corticotropin-releasing hormone (CRH), and the pathways
involved. Rats were
restrained or injected intraperitoneally with CRH or saline. Colonic
segments were mounted in Ussing chambers, in which ion secretion and
permeability (conductance and probe fluxes) were measured. To test the
pathways involved in CRH-induced changes, rats were pretreated with
hexamethonium, atropine, bretylium, doxantrazole, -helical
CRH-(9
41) (all intraperitoneally), or aminoglutethimide
(subcutaneously). Restraint stress increased colonic ion secretion and
permeability to ions, the bacterial peptide FMLP, and horseradish
peroxidase (HRP). These changes were prevented by
-helical
CRH-(9
41) and mimicked by CRH (50 µg/kg). CRH-induced changes in
ion secretion were abolished by
-helical CRH-(9
41), hexamethonium,
atropine, or doxantrazole. CRH-stimulated conductance was significantly
inhibited by
-helical CRH-(9
41), hexamethonium, bretylium, or
doxantrazole. CRH-induced enhancement of HRP flux was significantly
reduced by all drugs but aminoglutethimide. Peripheral CRH reproduced
stress-induced colonic epithelial pathophysiology via cholinergic and
adrenergic nerves and mast cells. Modulation of stress responses may be
relevant to the management of colonic disorders.
neuroimmune interactions; stress; colon; Wistar Kyoto rats
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INTRODUCTION |
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CLINICAL EVIDENCE INDICATES that life stress is associated with reactivation of colonic disorders (4, 8). However, the mechanism by which stress influences colonic pathophysiology remains obscure. Corticotropin-releasing hormone (CRH) has been implicated in various stress-induced abnormalities, including changes in both human and rat gastrointestinal functions such as motility and gastric acid secretion (19, 34).
Epithelial cells in the gastrointestinal tract have recently been recognized as critical cells in the regulation of immunoinflammatory disorders (10), with both an active role (production of cytokines and chemokines) and a passive role (acting as a barrier to limit the uptake of luminal antigens). However, relatively little information is available regarding the effects of stress and CRH on gastrointestinal epithelial physiology, particularly in the colon. It has been shown that physical and psychological stressors induce marked alterations in water and electrolyte transport across the epithelium in human and rat small intestine (2, 31, 32). In addition, epithelial permeability to small and large molecules has been reported to be enhanced by stress in the rat jejunum (17, 32). Although the colon is considered as a relatively tight epithelium for medium or large size molecules, permeation by the bacterial peptide FMLP (mol mass 438 kDa) and inulin (mol mass 5 kDa) does occur in the intact colon (21, 29). Factors that modulate tight junction resistance, such as prostaglandins, seem to be involved in this phenomenon, although the ultimate mechanism remains to be elucidated (21, 29). Increased transepithelial uptake of macromolecules, such as food or microbial-derived antigens, could provide a pathogenic link between intestinal barrier defects and immunologic abnormalities that may be involved in the initiation and/or relapses of mucosal inflammation in inflammatory bowel diseases, especially in stress-susceptible individuals (43).
On the basis of previous findings related to the role of CRH in stress-induced changes in gastrointestinal physiology (19, 34), we hypothesized that CRH might produce stresslike colonic epithelial abnormalities. The specific aims of the present study were to compare the effects of stress and peripheral CRH on colonic epithelial ion secretion and permeability to ions and macromolecules and to determine the mechanisms involved in the CRH-induced mucosal pathophysiology.
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METHODS |
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Animals
Male Wistar Kyoto rats (200-250 g) from Charles River Laboratories, (St. Constant, PQ, Canada), housed two per cage, were maintained on a normal 12:12-h dark-light cycle and provided with food and water ad libitum. Rats were handled daily by the same investigator for 2 wk before the study. The experimental procedures were performed at the same time of the day, between 8:00 and 10:00 AM, to minimize the effect of circadian rhythm. Rats were euthanized by decapitation. All procedures were approved by the Animal Care Committee at McMaster University.Drugs and Treatments
Stress protocol. Rats were placed in a Plexiglas adjustable restraining device (6 × 21.5 cm) for 2 h at 8°C, followed by 2 h of free movement in their home cage at 22°C. Control rats were maintained in their home cage.
Drugs.
CRH and -helical CRH-(9
41) (Peninsula Laboratories, Belmont, CA)
were dissolved according to the manufacturer's instructions, aliquoted, and kept frozen at
80°C until used. Immediately
before the experiments, the peptides were diluted in saline for
intraperitoneal injections. Doxantrazole, a gift from Burroughs
Wellcome (Research Triangle Park, NC), atropine sulfate, bretylium
tosylate, and hexamethonium (all from Sigma, St. Louis, MO) were
dissolved immediately before the experiments and administered
intraperitoneally in a 1-ml/kg volume injection.
Epithelial Colonic Measurements
Ussing chamber studies. The distal colon was removed, placed in 37°C oxygenated Krebs buffer, stripped of longitudinal muscle and myenteric plexus, and opened along the mesenteric border. Four adjacent pieces from each rat were mounted in Ussing chambers (W-P Instruments, Narco Scientific, Mississauga, ON, Canada). The chamber opening exposed 0.6 cm2 of tissue surface area to 8 ml of circulating oxygenated Krebs buffer at 37°C. The buffer contained (in mM) 115 NaCl, 1.25 CaCl2, 1.2 MgCl2, 2.0 KH2PO4, and 25 NaHCO3, pH 7.35. In addition, the serosal buffer also contained 10 mM glucose as an energy source that was 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 (W-P Instruments). Isc (µA/cm2) was recorded continuously by a computer connected to the voltage clamp system. Tissue conductance (G) was calculated according to Ohm's law and expressed as mS/cm2.
Mucosal-to-serosal transport of probes was determined by measuring transepithelial flux of FMLP and horseradish peroxidase (HRP). In some studies, radiolabeled [3H]FMLP (DuPont, Wilmington, DE) was added to the mucosal buffer (1 Ci/ml) and brought up to a concentration of 10Epithelial uptake of HRP. Epithelial uptake of HRP was measured in electron photomicrographs. Tissues were removed 2 h after the addition of HRP to the chambers, were fixed in 2.5% glutaraldehyde in 0.1 M sodium-cacodylate buffer (pH 7.4) for 2 h at 22°C, were rinsed for 18 h (4°C) with 0.05 Tris buffer (pH 7.6), and then were washed three times for 5 min. Methods for HRP product identification were modified from Graham and Karnovsky (12). Peroxidase activity was demonstrated by incubating the tissues for 15 min in 0.5 mg/ml diaminobenzidine in 0.05 M Tris buffer (pH 7.6, 22°C) and subsequently incubating for 15 min in the same buffer containing 0.01% H2O2. Tissues were then processed for routine transmission electron microscopy. Quantitative analysis was performed on coded high-magnification photomicrographs, 60 per group (3-4 tissues from each of 4 rats/group). The area of HRP-containing endosomes within colonic enterocytes was determined (with a computerized image analysis system) in the apical region (between the microvilli and the nucleus), the midregion (adjacent to the nucleus), and the basal region (between the nucleus and the basal membrane) and was reported as µm2/window (window area 25 × 30 µm2). Tissues were also examined for evidence of paracellular HRP transport.
Experimental Design
In the first part of the study, we determined the effects of stress on colonic Isc, G, and fluxes of FMLP and HRP and compared results with the effects induced by different doses of CRH (5, 12.5, 25, and 50 µg/kg ip). Pilot experiments showed that epithelial transport abnormalities were maximal at 4 h after injecting CRH or beginning the restraint stress. Baseline values for Isc, as an indicator of ion secretion, and G, as an indicator of ion permeability, were calculated at equilibrium, 15 min after mounting the tissues, and then every 30 min for 2 h. Fluxes of FMLP and HRP were determined at 30-min intervals over the course of the study and expressed as the average value of two consecutive stable flux periods.In the second part of the study, we evaluated whether peripheral CRH
contributed to the colonic stress responses. Rats were injected
intraperitoneally with either saline or -helical CRH-(9
41) (250 µg/kg), 30 min before the stress protocol. Ussing chamber studies
were conducted as described and colonic
Isc,
G, and HRP flux examined.
Finally, to characterize the mechanisms involved in the response to
intraperitoneal CRH, the same experimental protocol was followed. Thus
30 min before the administration of the higher dose of CRH (50 µg/kg;
maximal colonic responses) or its vehicle, different groups of rats
were treated with -helical CRH-(9
41) (250 µg/kg ip),
doxantrazole (20 mg/kg), hexamethonium (15 mg/kg), atropine sulfate (1 mg/kg), bretylium tosylate (15.8 mg/kg), or aminoglutethimide (300 mg/kg). The doses of
-helical CRH-(9
41), doxantrazole,
hexamethonium, atropine, and bretylium were based on previous studies
showing effective blocking of CRH receptors, mucosal mast cell
activation, or muscarinic, nicotinic, and adrenergic receptors, in
comparable experimental conditions (7, 36). Ussing chamber studies were
then conducted as previously described.
Statistical Analysis
Results are expressed as means ± SE. Multiple groups were compared by Dunnett's test and Fisher's exact test after a significant one-way ANOVA. Single comparisons were performed by paired or unpaired Student's t-test when appropriate. P values of <0.05 were considered significant. ![]() |
RESULTS |
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Comparison of Stress and CRH Effects on Colonic Epithelial Physiology
Stress induced a significant increase in both baseline Isc and G compared with controls. CRH (50 µg/kg) also induced significant increases in both baseline Isc and G compared with controls that were similar to those induced by stress (Table 1).
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Both stress and CRH (50 µg/kg) induced a two- to threefold increase
in HRP fluxes compared with their controls (Table 1). In addition,
tissues from stressed rats were twice as permeable to FMLP (1.04 ± 0.19 vs. 0.52 ± 0.13 µmol · h1 · cm
2,
P < 0.05) as tissues from control
rats (n = 6 rats/group).
The stimulatory effects of intraperitoneal CRH on rat colonic
Isc and
G were dose dependent (Table
2). The averaged percent increase in
Isc over vehicle
values was 36, 61, 64, and 96% for 5, 12.5, 25, and 50 µg/kg of CRH,
respectively. CRH also increased G
over vehicle values by 23, 20, 30, and 35% for 5, 12.5, 25, and 50 µg/kg of CRH, respectively. HRP flux was also enhanced in a
dose-dependent manner, ranging from a threefold increase at the highest
dose of CRH (14.4 ± 2.7 pmol · h1 · cm
2)
to no significant increase at the lower doses (Fig.
1), compared with animals injected with
vehicle (5.2 ± 0.6 pmol · h
1 · cm
2).
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Stress and CRH Enhanced Transepithelial Uptake of HRP
HRP was evident within endosomes in colonocytes from stress and CRH rats as well as in their controls (Fig. 2, A-C). However, endosomes appeared significantly more dense and numerous in stressed and CRH groups compared with controls, and the area of HRP-containing endosomes was significantly increased in all of the apical, mid, and basal regions of colonic enterocytes (Fig. 3). In addition, HRP was observed within tight junctions and paracellular spaces but only in tissues from stressed and CRH groups (Fig. 2D). This finding was present in every rat but not in all tissue sections.
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Pharmacological Modulation of Stress and CRH-Stimulated Colonic Isc, G, and HRP Flux
Treatment with
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Hexamethonium (15 mg/kg ip) or atropine (1 mg/kg ip) abolished the
CRH-stimulated increase in baseline
Isc, whereas
bretylium (15.8 mg/kg ip) showed no effect (Fig.
6). The increase in
G induced by CRH was reduced by
hexamethonium or bretylium but was not altered by atropine (Fig. 6).
The increase in HRP flux induced by CRH was reduced by hexamethonium,
atropine, or bretylium, all to a similar extent (from 14.4 ± 2.7 to
7.45 ± 2.4, 7.9 ± 1.8, and 8.0 ± 3.4 pmol · h1 · cm
2,
respectively, P < 0.05 vs. CRH). No
changes in epithelial parameters were observed when each drug was
administered in control rats.
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Treatment with doxantrazole (20 mg/kg) reduced the CRH-stimulated
increases in baseline
Isc,
G, and HRP flux by 74, 73, and 96%,
respectively (Fig. 7). In contrast,
aminoglutethimide did not change these parameters. Neither doxantrazole
nor aminoglutethimide changed
Isc,
G, and HRP when administered to
control rats.
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DISCUSSION |
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Our results show that stress stimulated distal colonic secretion,
indicated by increased
Isc, and
permeability, indicated by elevated G
and fluxes of FMLP and HRP. The effects of stress were due, at least in
part, to the action of CRH, because these responses were inhibited by
intraperitoneal administration of the CRH antagonist -helical
CRH-(9
41) and were mimicked by peripheral administration of CRH. Our
study indicates that the CRH effects were mediated by specific receptor
activation in the periphery because they were inhibited by
intraperitoneal administration of
-helical CRH-(9
41). Moreover,
the CRH-induced increases in Isc,
G, and HRP fluxes involved neural
pathways and mast cells as indicated by modulation of the responses by
hexamethonium, atropine, bretylium, and doxantrazole. Conversely, the
hypothalamic-pituitary-adrenal axis, through the release of steroids,
was not implicated in the stimulatory effects of CRH, shown by the lack
of effect of aminoglutethimide on these responses.
In this study, we have extended our previous findings (17, 32) to show that stress-induced pathophysiology occurs not only in the jejunum but also in the distal colon. Studies from other laboratories have documented stress-induced increases in mucus production (5), transit (34), and fluid secretion in rat colon (9). These responses, along with the stress-induced increases in ion secretion reported in this study, indicate that stress may contribute to host defense by flushing noxious material out of the lumen. However, other effects of stress may be detrimental, and, in particular, stress-induced epithelial responses may be involved in the initiation or potentiation of relapses of inflammatory bowel disease. Several reports suggest that intestinal permeability is increased in inflammatory bowel disease patients (25, 43); increased uptake of food or microbial antigens may activate resident immune cells, leading to an inflammatory response. Our study shows that colonic epithelium from stressed rats was significantly more permeable to bioactive molecules such as the bacterial proinflammatory peptide FMLP. Moreover, the stress-induced barrier defect extended to macromolecular proteins with antigenic properties such as HRP (mol mass 44 kDa). We previously showed that stress caused a dramatic increase in the flux of HRP across jejunal epithelium by stimulating transport via both intracellular and paracellular pathways (17). Here, we showed that the colon also responds to stress with enhanced transepithelial protein transport via transcellular and paracellular routes through tight junctions.
Peripheral administration of CRH has been shown to mediate or induce
stresslike responses in the gastrointestinal tract, including decreased
gastric acid secretion (19), increased duodenal bicarbonate secretion
(19), and motility changes (34). The direct effects of CRH are mediated
by the activation of specific receptors on the target cell population
(37). In our study, intraperitoneal CRH increased
Isc,
G, and HRP permeability, and these
responses were inhibited by intraperitoneal -helical CRH-(9
41).
Because both
-helical CRH-(9
41) and CRH are believed not to cross
the blood-brain barrier from the periphery to the brain (24), our findings indicate that colonic abnormalities induced by CRH were mediated by peripheral CRH receptors. At least two different subtypes of CRH receptors have been described in the periphery,
CRH-R1 and
CRH-R2
(37). Our work did not
identify the population of receptors involved. More specific agonists
and/or antagonists for CRH receptor subtypes will be
required to address this particular issue. The importance of peripheral
CRH receptors in stress responses in the intestinal tract is
highlighted in our study because the effects of stress on colonic
physiology were also inhibited by intraperitoneal
-helical
CRH(9
41).
Colonic secretion and permeability are regulated by neural and immune factors. In the present study, the increase in Isc induced by CRH was abolished by both the muscarinic antagonist atropine and by the nicotinic antagonist hexamethonium but was not affected by the adrenergic blocker bretylium. Previous studies have shown that muscarinic receptors on epithelial cells and enteric neurons in rat colon accounted for 70% of the Isc increase in response to cholinergic receptors, whereas the remaining 30% was mediated by nicotinic receptors on enteric neurons (7, 27). These studies, in conjunction with the present report, suggest that the CRH-induced stimulation of colonic Isc may have been mediated by the release of acetylcholine.
CRH also increased colonic G, indicating enhanced permeability to ions. This increase was blocked by hexamethonium and bretylium but not affected by atropine, suggesting that the effects of CRH on colonic G are mediated by both nicotinic and adrenergic receptors. We showed that the CRH-stimulated increase of HRP flux was reduced by muscarinic, nicotinic, and adrenergic antagonists. Cholinergic mechanisms have been implicated previously in macromolecular transport because stress-enhanced permeability to HRP in the rat jejunum was reduced by atropine (17), and other studies have shown that cholinergic agonists and vagal stimulation increased intestinal epithelial permeability (28, 30). A recent report indicated that paracellular permeability to Evans blue (mol mass 961 kDa) was regulated by adrenergic mechanisms (18). Our results, along with those from previous studies, suggest a complex interplay between sympathetic and parasympathetic nerves in the regulation of colonic permeability.
We also showed that the CRH-stimulated responses in colonic epithelium were dependent on mast cell activation because they were abolished by the mast cell stabilizer doxantrazole. Mast cells contain mediators, such as prostaglandins, histamine, and serotonin, that directly alter epithelial transport properties. Moreover, other stress-induced epithelial responses, such as colonic mucin release, were absent in mast cell-deficient mice, whereas reconstitution of bone marrow with mast cells reversed that response to normal stress values (6). Previous reports showing mast cell proximity to CRH-like nerves in the brain (36), expression of CRH-R1 on cultured human leukemic mast cells (35), and degranulation of mast cells in the skin and colon with parallel increases in vascular permeability and mucin secretion, respectively, after peripheral administration of CRH (5, 35) support the hypothesis that mast cells could have been activated directly by CRH. However, colonic mast cells could have been activated indirectly by neuropeptides released from CRH-stimulated neural processes. This view is supported by studies showing close proximity between peptidergic terminals and mast cells in the rat intestinal mucosa (33), degranulation of mast cells by substance P in the intestine (14), and a functional interaction between intestinal mast cells and neuropeptide-containing nerves reflected by a mast cell-mediated increase in intestinal ion secretion on substance P administration (41). Despite the nature of the mechanism of mast cell activation, our study illustrates that cross talk between neural and immune cells is involved in the regulation of epithelial transport functions in the colon.
Peripheral CRH stimulates corticotropin release that ultimately causes adrenal steroid secretion (37). Because both mineralcorticoids and glucocorticoids participate in the regulation of ion transport in the rat distal colon (3), we blocked their synthesis with aminoglutethimide, to test the role of CRH-induced steroid release in the observed colonic abnormalities. Aminoglutethimide did not alter basal or CRH-stimulated colonic responses, making it unlikely that steroids contributed to these effects.
Our study raises some interesting questions that remain to be answered, including the cellular origin of CRH in the periphery and its site of action. In the colon, CRH mRNA has been detected in the vicinity of the base of the crypts, probably being produced by enterochromaffin cells (16). CRH-like immunoreactivity has also been described in myofibroblasts, autonomic ganglia, and intrinsic and extrinsic neural cells (20, 26, 33, 40, 42). Furthermore, CRH is synthethized by several immunocytes on appropriate stimulation (15). Finally, colonic enterocytes themselves may produce CRH, because their ability to secrete CRH and other regulatory molecules has been reported (39). Because these elements are represented in the intestinal wall, this group of studies, in conjunction with ours, suggests that local release of CRH in the gastrointestinal tract could play an important role in the modulation of epithelial physiology.
Other than mast cells, potential targets for CRH in the periphery may include other immune cells, as well as neural, epithelial, or endothelial cells. These cells might be partially responsible for some of the CRH-induced changes in epithelial physiology. Receptors for CRH have been demonstrated by molecular analysis in the intestinal wall (20). Binding and pharmacological studies have also provided evidence for CRH receptors on several immune cells and intrinsic and extrinsic neurons (1, 13, 15, 36, 38). The presence of CRH receptors on colonocytes is presently unresolved, although the requirement of mast cell activation for the full expression of CRH-induced changes in colonic physiology in our study suggests that those receptors are unlikely in colonic epithelium.
In conclusion, we have demonstrated that stress induced marked alterations in colonic epithelial physiology via CRH-mediated pathways involving mast cells and nerves. Recognition of the potentially adverse effects of stress in the gastrointestinal tract may lead to novel preventive and therapeutic approaches in colonic functional and inflammatory disorders.
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ACKNOWLEDGEMENTS |
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We acknowledge the expert technical assistance of M. Benjamin and the staff of the Electron Microscopy Unit, Faculty of Health Sciences, McMaster University.
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FOOTNOTES |
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This study was supported by grants from the Medical Research Council of Canada, the Academia de Ciencias Medicas de Catalunya i Balears, and the Sociedad Catalana de Digestología (1996-97). J. Santos was a recipient of a Medical Research Council/Pharmaceutical Manufacturers' Association of Canada (Astra Research Initiative) Fellowship. D. Yates was a recipient of a Canadian Association of Gastroenterology/Hoescht Marion Roussel Summer Student Scholarship.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. H. Perdue, Intestinal Disease Research Program, HSC-3N5C, McMaster Univ., 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (E-mail: perdue{at}fhs.mcmaster.ca).
Received 16 March 1999; accepted in final form 12 May 1999.
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