Molecular Psychiatry (2002) 7, 474–483
 2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00
www.nature.com/mp
ORIGINAL RESEARCH ARTICLE
Marked suppression of gastric ulcerogenesis and
intestinal responses to stress by a novel class of drugs
KE Gabry1, GP Chrousos2, KC Rice3, RM Mostafa4, E Sternberg1, AB Negrao1,
EL Webster1, SM McCann5 and PW Gold1
1
Clinical Neuroendocrinology Branch, National Institute of Mental Health, Intramural Research Program, National Institutes
of Health, Bethesda, MD, USA; 2Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and
Human Development, Intramural Research Program, National Institutes of Health, Bethesda, MD, USA; 3Laboratory of
Medicinal Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, Intramural Research Program,
National Institutes of Health, Bethesda, MD, USA; 4Department of Surgery, University of Pittsburgh Medical Center,
Pittsburgh, PA, USA; 5Pennington Biomedical Research Laboratories, Louisiana State University, Baton Rouge, LA, USA
When exposed to prolonged stress, rats develop gastric ulceration, enhanced colon motility
with depletion of its mucin content and signs of physiological and behavioral arousal. In this
model, we tested whether antidepressants (fluoxetine and bupropion), anxiolytics (diazepam
and buspirone) or the novel nonpeptide corticotropin-releasing hormone (CRH) type-1 receptor (CRH-R1) antagonist, antalarmin, modify these responses. Fluoxetine, bupropion,
diazepam and antalarmin all suppressed stress-induced gastric ulceration in male Sprague–
Dawley rats exposed to four hours of plain immobilization. Antalarmin produced the most
pronounced anti-ulcer effect and additionally suppressed the stress-induced colonic hypermotility, mucin depletion, autonomic hyperarousal and struggling behavior. Intraperitoneal
CRH administration reproduced the intestinal but not the gastric responses to stress while
vagotomy antagonized the stress-induced gastric ulceration but not the intestinal responses.
We conclude that brain CRH-R1 and vagal pathways are essential for gastric ulceration to
occur in response to stress and that peripheral CRH-R1 mediates colonic hypermotility and
mucin depletion in this model. Nonpeptide CRH-R1 antagonists may therefore be prophylactic
against stress ulcer in the critically ill and therapeutic for other pathogenetically related gastrointestinal disorders such as peptic ulcer disease and irritable bowel syndrome.
Molecular Psychiatry (2002) 7, 474–483. doi:10.1038/sj.mp.4001031
Keywords: functional gastrointestinal disorders; corticotropin-releasing; type 1 receptor; peptic ulcer;
irritable bowel; nonpeptide; antagonist
Introduction
In addition to predisposing to mental illness and altering the endocrine milieu of the organism, stress frequently precipitates marked disturbance of visceral
functions. The gastrointestinal tract is one of the most
vulnerable viscera to aversive psychological stimuli.1
Individual variability in responding to stress ranges
from nausea and vomiting to abdominal pain and
diarrhea. Severe stress of life-threatening conditions,
eg shock, head trauma, myocardial infarction, may be
associated with stress ulcer (SU) development; a potentially fatal condition whose pathophysiology is still
obscure.2 Less severe, prolonged stress has been implicated in the pathogenesis of peptic ulcer disease3 and
irritable bowel syndrome (IBS).4 These disorders
Correspondence: KE Habib, MD, PhD, Clinical Neuroendocrinology branch, IRP, NIMH, NIH Bldg 10, Rm 2D-46, Bethesda, MD
20892-11284, USA. E-mail: habib얀codon.nih.gov
Received 22 May 2001; revised 24 September 2001; accepted 13
October 2001
appear to be interrelated and to be associated with
affective and anxiety disorders.5,6 Moreover, early life
adversity is associated with more prevalence of mood
and anxiety disorders7,8 as well as predisposition to
gastric ulceration9 and IBS10 later in adulthood. In spite
of the fact that these stress-related gastrointestinal (GI)
disorders constitute the most common presentation in
gastroenterology and primary care clinics,11 our ability
to treat them is still limited as their underlying pathophysiologic mechanisms remain to be deconvoluted.12
Since its identification as a classic component of the
stress response by Selye (1936)1 and establishing it as
a model of somatic consequences of stress by Brodie,13
stress-induced gastric ulceration has been widely used
in studying visceral responses to stress. This includes
exploring protective mechanisms against stress by
measuring their potential for preventing stress ulceration. The same model is also useful as a model of nonulcer dyspepsia and peptic ulcer, particularly in the
context of their biopsychosocial nature, in man.3,14
Many researchers in the area of gastrointestinal drugs
screen their new compounds in the stress-induced gas-
Marked suppression of gastric ulcerogenesis
KE Gabry et al
tric ulcer model.15 Even though for more than a decade
the focus of the etiology of peptic ulcer shifted to helicobacter infection, many clinicians now believe that
the role of Helicobacter pylori in the causation of peptic ulcer is no more than a partial one.3,16 Other equally
important factors include genetic makeup and vulnerability to stress.17–19 Moreover, a recent study has
negated the involvement of H. pylori in stress ulcer
bleeding in critically ill patients.2
The pivotal role of the brain in the pathogenesis of
stress-induced gastric mucosal damage has been well
established. Increased vulnerability to depression20
and anxiety21 in experimental animals is paralleled
with ulcer development. The same holds true for
humans.5,22 Moreover, classic antidepressants23,24 and
anxiolytics25,26 significantly reduce stress ulcer formation, perhaps to a greater extent than that seen with
traditional therapies such as cimetidine and antacids.27
Indeed, induction of gastric lesions in rodents by sociopsychological stress is useful as an experimental model
to assess the anxiolytic activity of novel drugs and the
anxiolytic potency of a drug may be predicted by its
anti-ulcer effect.28
While the role of corticotropin-releasing hormone
(CRH) in mediating the stress-induced alteration of gastrointestinal secretory and motor responses is fairly
established,29–31 its role in the pathogenesis of stress
ulcer has been debatable.32,33 CRH is a 41-amino acid
peptide that was first isolated as the principal hypothalamic hormone that mobilizes the pituitary-adrenal
responses to stress34 and subsequently identified as a
key player in the behavioral and autonomic responses
to stress in rodents35 and nonhuman primates.36 Several studies have shown that both stress and central
CRH administration decrease gastric acid secretion,
gastric emptying, and small bowel transit while markedly enhancing large bowel transit.29,37 Moreover,
immobilization stress of rats causes increased colonic
mucin and prostaglandin E2 secretion, increased
colonic mucosal levels of cyclooxygenase-2 mRNA,
and degranulation of colonic mast cells, which are
antagonized by alpha-helical CRH 9–41, a non specific
peptide CRH antagonist.31 Central administration of
peptide CRH receptor antagonists prevents the gastrointestinal secretory and motor responses elicited by
either stress or exogenous CRH in a dose-dependent
fashion.30,38
The clinical development of nonpeptide antagonists
which cross the blood–brain barrier to reset brain CRHR1 for the treatment of anxiety40 and melancholic
depression41 is currently the focus of extensive
research. We have recently reported that oral administration of antalarmin, a member of this novel family of
drugs, attenuates neuroendocrine and behavioral
responses to stress in primates.36 Due to the significant
overlap in the neural circuitry that mediates mental
and gastrointestinal responses to stress,39 we hypothesized that CRH-R1 plays a role in the pathogenesis of
gastrointestinal injury and/or dysfunction during
stress. The recent availability of non-peptide CRH-R1
antagonists enabled us to examine the role of CRH-R1
in an animal model that simultaneously reproduces
features of stress ulcer13 and IBS.4 Antalarmin, N-butylN-ethyl-[2,5,6-trimethyl-7-(2,4,6 trimethylphenyl)-7Hpyrrolo[2,3-d]pyrimidin-4-yl] amine, is a selective
CRH-R1 antagonist that penetrates the blood–brain barrier and suppresses neuroendocrine, autonomic and
behavioral responses to stress in primates.36 Other pyrrolopyrimidine compounds also exert anxiolytic
effects42 and suppress colon hypermotility43 in rodents.
The major advantage of nonpeptide antagonists is their
potential clinical utility in humans.44 As opposed to
peptide antagonists that require direct injections into
the brain to access central CRH receptors, antalarmin
can neutralize CRH-R1 whether in the brain or in the
periphery after oral administration.36
Here we first conducted a dose-response study to
examine the effects of antalarmin on stress-induced
gastric ulceration in adult male Sprague–Dawley rats.
To determine whether antalarmin’s effects are due to
moderating the activity of the stress neurocircuitry, we
tested it against other known antidepressants
(bupropion and fluoxetine) and anxiolytics (diazepam
and buspirone). We chose the latter drugs for their nonclassic antidepressant effects and lesser anticholinergic
activity.45 In addition to scoring gastric ulceration, we
evaluated colon specimens for mucin depletion and
counted the stool pellets as an index of enhanced colon
motility in response to stress.46 Stress-induced
enhancement of intestinal transit in the rat has been
proposed as an animal model for irritable bowel syndrome.4 Moreover, defecation scores have been long
known as valid representatives of fearfulness and anxiety in rodents.47 Plasma catecholamines and struggling
behavior were included as indices of autonomic and
behavioral arousal in this model.
475
Experimental procedures
Drugs
Antalarmin, N-butyl-N-ethyl-[2,5,6-trimethyl-7-(2,4,6 trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl] amine,
was synthesized in our laboratory as previously
described.48 For intraperitoneal (IP) injections, antalarmin was dissolved in cremophor/ ethanol/ saline,
5:5:90 by volume, and injected (0.5 ml per animal)
immediately after preparation. Diazepam (Roche Lab,
Nutley, NJ, USA) was suspended in 0.5% carboxymethylcellulose and injected intramuscularly.49 Buspirone (Sigma, St Louis, MO, USA), bupropion (Sigma)
and fluoxetine (Tocris Cookson, Ballwin, MO, USA)
were dissolved in saline, and all were injected as 0.5
ml per animal IP. For the dose response experiment,
four animal groups were included into which antalarmin was injected at doses of 0, 15, 30 and 45 mg kg−1.
For the comparative study, six treatment groups were
included: saline (0.5 ml rat−1), antalarmin (20 mg kg−1),
diazepam (5 mg kg−1),50 buspirone (4 mg kg−1),51 bupropion (30 mg kg),52 and fluoxetine (10 mg kg−1).53 CRH
(human/rat CRF; Peninsula Lab, San Carlos, CA, USA)
was dissolved in saline and injected IP (0.5 ml rat−1) at
a single dose of 5 ␮g kg−1 in freely moving animals 4 h
prior to their killing.
Molecular Psychiatry
Marked suppression of gastric ulcerogenesis
KE Gabry et al
476
Animal experiments
Adult male (200–250 g) Sprague–Dawley rats (Taconic
Farms, Germantown, NY, USA) were housed under
controlled conditions on a 12-h light/12-h dark cycle
for an average of 1 week after arrival to the animal
facility. Rats were housed 3 to a cage and provided
with food and water ad libitum except for the last 24 h
preceding the experiment, when they were individually housed in mesh bottom cages and deprived of
food, but not water. In our experience, fasted rats
develop stress ulcers more readily and consistently
than non-fasted subjects, particularly if stress is
applied in the afternoon. All experiments were started
between the hours of 12:00 and 13:00 to minimize the
influence of circadian rhythm on the parameters under
investigation. Animal procedures were approved by
the National Institute of Child Health and Human
Development Animal Care and Use Committee and
conducted with strict adherence to the National Institutes of Health Guidelines of Animal Use in Intramural Research.
Animals groups received half the dose of their drug
intraperitoneally (IP) twice at −2 h and +2 h of starting
immobilization in the dose response experiment. A
pilot experiment showed better anti-ulcer responses if
the drugs were administered in full doses twice at −24
and −2 h prior to immobilization (data not shown), and
therefore this latter regimen was used for the comparative study. Subdiaphragmatic or sham vagotomy were
performed 4 days prior to the experiment as previously described.54,55
Immobilization stress lasted for 4 h and was performed essentially as described by Brodie13 with slight
modification. Briefly, awake rats were placed in the
prone position on specifically designed stainless steel
plates equipped with semi-circular rings that prevent
the animal from turning around to access its taped
lower trunk and feet. The animal’s struggling behavior
was blindly scored for 5 min at the summits of immobilization hours 1, 2 and 3, according to a previously
validated scale that scores one point for every act of
jaw movement, body rolling, tail flipping or vocalization, with simultaneous acts counted as the sum of
their individual components.49 The three time point
results were averaged and expressed as the struggling
score per hour. Colonic motility was estimated by
counting the number of fecal pellets expelled during
the immobilization period.29,46
After the elapse of 4 h, rats were released from their
restrainers and immediately guillotined. Trunk blood
was collected in ice-chilled tubes and spun in a cooling
centrifuge at 1000 × g to obtain plasma for the assay of
epinephrine and norepinephrine by high performance
liquid chromatography coupled with electrochemical
detection as previously described.36 A sagittal laparotomy was performed to collect stomachs and colonic
segments 10 cm proximal to the anal orifice. The latter
specimens were fixed in formalin, embedded in paraffin and stained with hematoxylin, eosin and alcian
blue for histological estimation of mucin content.
Briefly, 7-␮m sections perpendicular through the
Molecular Psychiatry
mucosa in an area containing 10 parallel aligned crypts
were examined for the percentage of mucin-positive
goblet cells to the total number of colonocytes lining
the superficial half of the crypts epithelium as
described previously.46 Stomachs were washed in ice
cold saline and opened along the greater curvature to
wash the interior with phosphate buffered saline for
scoring gastric ulcers as the sum of their cumulative
length in mm per stomach with the aid of a magnifying
lens and a micrometer.56 All samples were coded and
blindly scored. Because of the protective effect of food
against SU, stomachs containing food remnants were
excluded regardless of their ulcer scores.
Statistical analysis
ANOVA followed by post-hoc (Dunnett’s Multiple
Comparison) test was used to study the effects of different treatments on plasma catecholamines, stress ulcer,
intestinal mucin, struggling and defecation scores.
ANOVA post-test for linear trend was used to express
the results of the dose-response study. On all
occasions, a P level was considered statistically significant at two-tailed values of 0.05 or less. Statistical
analysis and graphic presentations were aided by
Prism version 3.0 for Macintosh (GraphPad, Ithaca,
NY, USA).
Results
Figures 1a and 1b show the dose response effects of
antalarmin on stress-induced gastric ulceration (SU).
Antalarmin reduced SU scores in a dose-dependent
fashion, namely from 12.63 ± 1.711 at 0 mg kg−1 to
2.000 ± 1.323 at 45 mg kg−1. ANOVA post-test for linear
trend showed a slope of −1.788 (R2 = 0.539, P ⬍0.001).
In freely moving animals, none of the drugs significantly caused any significant change in gastric ulceration, colonic mucin or defecation scores as compared
to saline treatment (data not shown), and therefore the
effects of different drugs on the parameters under
investigation during stress are only presented.
In saline-treated animals, exposure to immobilization stress precipitated gastric ulceration (from 0.125
± 0.125 mm to 15.88 ± 2.463 mm, t = 5.41, P ⬍ 0.001),
colonic mucin depletion (from 13.4 ± 2.73 to 4.625 ±
0.944, t = 3.03, P ⬍ 0.01), and elevation of plasma epinephrine (from 8.40 ± 2.22 to 21.11 ± 2.55 ng ml−1, t =
3.76, P ⬍ 0.01 ) and norepinephrine (from 5.45 ± 1.278
to 10.23 ± 0.849 ng ml−1, t = 3.243, P ⬍ 0.01) as compared to freely moving animals.
There were no significant differences between saline
treatment and any of the vehicles used for drug dissolution during stress on stress ulceration, defecation
scores, struggling, colonic mucin, plasma epinephrine
or plasma norepinephrine (Table 1), and therefore we
used saline treatment as the control group.
The ability of antalarmin and other psychotropic
drugs to reduce stress ulcer are shown in Figures 2a
and 2b. Significant reductions of stress ulceration from
15.88 ± 2.463 mm were achieved by antalarmin (to
1.392 ± 0.664 mm, P ⬍ 0.01) followed by diazepam (to
1.833 ± 0.726 mm, P ⬍ 0.01), bupropion (to 4.429 ±
Marked suppression of gastric ulcerogenesis
KE Gabry et al
Table 1 Effects of different vehicles used for drug dissolution on the stress-induced gastric ulceration, colon mucin, stool
pellet output, struggling behavior and plasma epinephrine and norepinephrine
Gastric ulceration
Colon mucin
Pellet output
Struggling
Plasma epinephrine
Plasma norepinephrine
Saline
Vehicle A
Vehicle B
15.88 ± 2.463
4.625 ± 0.944
12.13 ± 1.663
25 ± 3.525
21.11 ± 2.55
10.23 ± 0.849
12.5 ± 3.212
4.4 ± 1.077
6.857 ± 1.471
24 ± 3.386
20 ± 5.33
15.2 ± 5.44
11.17 ± 3.24
4.333 ± 1.526
8.833 ± 2.4
24.2 ± 4.164
20.2 ± 4.091
19.4 ± 6.071
477
ANOVA comparison
F(2,20)
F(2,18)
F(2,18)
F(2,18)
F(2,15)
F(2,17)
=
=
=
=
=
=
0.77,
0.022,
2.292,
0.02,
0.022,
1.794,
P
P
P
P
P
P
=
=
=
=
=
=
0.476
0.978
0.13
0.978
0.98
0.196
Vehicle A used to dissolve antalarmin consisted of cremophor/ethanol/saline, 5:5:90 by volume, and injected (0.5 ml per
animal) intraperitoneally. Vehicle B used for diazepam consisted of 0.5% carboxymethylcellulose and injected intramuscularly
(0.5 ml per animal). The rest of the drugs were dissolved in saline and injected intraperitoneally (0.5 ml per animal). As
apparent in the ANOVA comparison column, there were no significant differences among the different vehicles on any of the
parameters under investigation.
1.706 mm, P ⬍ 0.01) and fluoxetine (to 7.438 ± 1.668
mm, P ⬍ 0.01). Buspirone did not significantly alter
SU scores (to 11.36 ± 2.497 mm, P ⬎ 0.05).
Both diazepam and antalarmin significantly
inhibited bowel hypermotility, as indicated by the
stool pellet output (from 12.13 ± 1.663 to 5.875 ± 1.072
SPO per 3 h, P ⬍ 0.05 by diazepam, and to 5.938 ±
0.972, P ⬍ 0.05 by antalarmin) (Figure 3a), but only
antalarmin (Figure 3b) significantly increased colonic
mucin during exposure to stress (from 4.625 ± 0.944 to
9.5 ± 1.195 mucin-positive colonocyte per 100
colonocytes, P ⬍ 0.01).
Figures 4a, 4b and 4c show the effects of antalarmin
and other psychotropic medications on struggling
scores, plasma epinephrine and norepinephrine,
respectively. As demonstrated in Figure 4a, antalarmin
(20 mg kg−1) was as effective as diazepam (5 mg kg−1)
in decreasing the score of struggling; from 25.0 ± 3.525
to 9.0 ± 2.591 (P ⬍ 0.001) and to 11.5 ± 3.065, P ⬍ 0.05),
respectively. Buspirone (15.5 ± 3.157), bupropion (17.5
± 3.541) and fluoxetine (30.0 ± 3.117) did not significantly reduce struggling behavior in this model (P ⬎
0.05). Reductions of plasma epinephrine from 21.11 ±
2.55 ng ml−1 were achieved by buspirone (10.79 ± 0.651
ng ml−1, P ⬍ 0.001) followed by diazepam (12.38 ±
1.143 ng ml−1, P ⬍ 0.001), then antalarmin (13.45 ±
1.178 ng ml−1, P ⬍ 0.05). Least changes were obtained
with the antidepressants bupropion (14.35 ± 2.18 ng
ml−1, P ⬎ 0.05) and fluoxetine (17.20 ± 2.31 ng ml−1, P
⬎ 0.05). Plasma norepinephrine (10.23 ± 0.849 ng ml−1)
significantly decreased by diazepam (4.168 ± 1.052 ng
ml−1, P ⬍ 0.01) followed by buspirone (6.29 ± 0.544 ng
ml−1, P ⬍ 0.05), then antalarmin (6.916 ± 0.463 ng ml−1,
P ⬍ 0.05). Non-significant changes were obtained with
the antidepressants bupropion (9.532 ± 2.56 ng ml−1, P
⬎ 0.05) and fluoxetine (13.22 ± 1.277 ng ml−1, P ⬎
0.05).
Compared to the saline-treated animals, intraperitoneal CRH did not significantly change the animals’
stress ulcer scores in non-stressed subjects (from 0.167
± 0.167 to 0.167 ± 0.167, t = 0.0, P = 0.50). However, it
significantly resulted in increased colon motility (from
4.3 ± 0.925 to 9.4 ± 1.75 SPO per 3 h, t = 2.577, P ⬍
0.05) (Figure 5a) and mucin depletion (from 7.89 ± 1.07
to 3.98 ± 1.06 mucin-positive colonocyte per 100
colonocytes, t = 2.596, P ⬍ 0.05) (Figure 5b). The effects
of intraperitoneal CRH on stool pellet output were also
abolished by antalarmin pretreatment (compared to the
vehicle group, t = 0.068, P ⬎ 0.05), but were marginally
affected by vagotomy or sham operation (compared to
the vehicle group, t = 2.146 and 2.26, P = 0.07 and P =
0.06, respectively) (Figure 5a). The effects of intraperitoneal CRH on mucin content were abolished by antalarmin pretreatment (compared to the vehicle group, t
= 0.546, P ⬎ 0.05) but were not significantly affected
by vagotomy or sham operation (compared to the
vehicle group, t = 4.181 and 3.06, P ⬍ 0.01 and P ⬍
0.02, respectively) (Figure 5b).
Figure 6 shows that compared to sham-operated rats,
vagotomized animals expressed significantly lower SU
scores in response to the same stressor (from 17.65 ±
3.85 to 3.86 ± 2.5, t = 3.048, P ⬍ 0.02), but their scores
of stool pellet output and colonocyte mucin content
were not statistically different (data not shown). CRH
administration had no effect on SU in either shamoperated (t = 1.459, P ⬎ 0.05) or vagotomized subjects
(t = 0.619, P ⬎ 0.05). On the other hand, antalarmin
pretreatment further attenuated SU in the sham-operated
animals (t = 3.473 and 1.17, P ⬍0.01 and ⬎ 0.05, for the
sham-operated and vagotomized rats respectively).
Discussion
Here we show that antalarmin not only reduced gastric
ulcerogenesis in responses to stress in a dose-dependent fashion, but also attenuated autonomic and
behavioral indices of arousal. The relatively new antidepressants, bupropion and fluoxetine, as well as
diazepam also exerted significant effects in attenuating
SU, supporting the notion that stress ulceration is a
centrally mediated process57 and that it is their psychotropic properties that contribute to their ulcerolytic
effects. We used the relatively new antidepressants
bupropion and fluoxetine that are virtually devoid of
antihistaminic or anticholinergic actions to avoid the
argument that antidepressants attenuated stress ulcerMolecular Psychiatry
Marked suppression of gastric ulcerogenesis
KE Gabry et al
478
ogenesis via blocking cholinergic or histaminergic
receptors.45 It is unlikely that antalarmin produced its
protective effects by merely attenuating glucocorticoid
responses to stress because we have previously found
that metyrapone does not modify the severity of stress
ulceration, at doses (40 mg kg−1 IM) that effectively
interfere with glucocorticoid synthesis in the same
model.58
Figure 1 (a) Dose response (0, 15, 30, 45 mg kg−1 IP) effects
of antalarmin on immobilization stress-induced gatric ulceration (SU) and (b) a photograph showing the interior of the
rats’ stomachs after the same treatment regimen. Fasted adult
male Sprague–Dawley rats (n = 8/group) received half the
dose of their drug intraperitoneally twice at −2 h and +2 h of
exposure to immobilization stress for 4 h, followed by
immediate termination and collection of trunk blood and stomachs. The latter were opened along the greater curvature
and the sum of each stomach ulcer length (cumulative ulcer
length) was measured with the aid of a magnifying lens and
a micrometer.
Molecular Psychiatry
The notion that stress ulceration is a centrally
mediated phenomenon is further supported by the failure of peripherally administered CRH to produce SUlike lesions in non-stressed subjects. On the contrary,
IP CRH reproduced colonic responses to stress; an
effect that was abolished by antalarmin pretreatment,
which is in line with previous studies.29,30,59,60 Moreover, we have observed that the effects of IP CRH were
not significantly different between vagotomized and
sham-vagotomized animals, which further supports the
notion that the intestinal effects of IP CRH were
mediated by peripheral mechanisms.
In spite of its known implication in virtually the
entire cascade of the stress response36 and the reproducibility of stress ulceration as a central component
of the animals’ responses to stress,1 the role of CRH in
the pathophysiology of stress ulcer has been controversial,3,61 with some studies reporting an ulcerogenic
effect of centrally administered CRH33 and others
reporting attenuation of cold restraint-induced gastric
mucosal lesions by the same treatment.32,62 The disagreement of our findings with the latter reports may
be due to the different techniques utilized and their
direct injections into the brain of excessive quantities
of CRH (in the microgram range) considering its naturally occurring concentrations (being in the picogram
range).36 At such concentrations, CRH acts locally as a
proinflammatory agent and to increase the
permeability of physiological barriers; both factors can
significantly confound the roles attributed to the
endogenous peptide.63
Because our focus is on stress ulceration, whose
pathophysiology cannot be simply explained by the
associated secretory and motor patterns,55,58 we chose
not to collect data on gastric secretion and motility.
Thus we avoided the trauma of laparotomy usually
required to collect gastric secretion or contractility records. Moreover, the ability of antalarmin to cross the
blood–brain barrier allowed us to avoid inflicting the
trauma of intracerebroventricular injections to the animals. The efficacy of antalarmin to antagonize stress
ulceration under these circumstances indicates that
endogenous CRH, via its type-1 receptor, mediates gastric ulcerogenesis caused mostly by plain immobilization, rather than other confounding physical stressors.
Moreover, such findings indicate the potential clinical
utility of nonpeptide CRH antagonists for the treatment
of stress ulcer in the critically ill and perhaps peptic
ulcer disease and non-ulcer dyspepsia.
Vagotomy produced antalarmin-like ulcerolytic
effects, which agrees with previous reports.64 The
vagus has been long implicated in mediating gastric
damage in response to stress.65 By virtue of its extensive projections to brain nuclei that participate in the
neurocircuitry of stress, the vagal efferents are well
positioned to mediate the gastric responses to stress.66
Indeed, the stomach receives by far more vagal innervation (both afferent and efferent) than any other
viscus.67 Efferent vagal fibers contain a variety of
neurotransmitters that could be ulcerogenic, eg TRH,
serotonin and opiate peptides.65,68 Moreover, the vagus
Marked suppression of gastric ulcerogenesis
KE Gabry et al
is essential for the maintenance of mast cells and enterochromaffin-like cells in the stomach.54 The latter cells
contain highly ulcerogenic substances as well. CRH is
extensively distributed in the hypothalamus and limbic system nuclei that project to the dorsal motor
nucleus of the vagus (DMV) and CRH containing nerve
fibers are found in the DMV.66,67
The effects of antalarmin in attenuating colonic
responses to stress were also marked. It significantly
suppressed both colonic hypermotility and mucin
depletion. This agrees with recently published studies
that have utilized different paradigms43,46 Moreover,
exogenous parenteral CRH reproduced the intestinal
responses to stress, in agreement with previous studies.30 This is of particular interest in the light of Fukudo’s finding of induction of colon motility in both IBS
patients and control subjects by CRH administered
intravenously, with significantly greater motility
indexes in IBS patients than in controls. Abdominal
symptoms evoked by CRH in IBS patients lasted significantly longer than those in the controls in the same
study.69 In the light of Williams’ hypothesis of the usefulness of enhanced bowel motility in response to
restraint stress to model an important aspect of IBS,
our findings here might suggest the potential clinical
utility of CRH-R1 antagonists to combat IBS.4
Vagotomy—on the other hand—did not alter the
intestinal responses to stress in this paradigm, again
suggesting that the site of antalarmin action in modulating colonic responses to stress is primarily peripheral. In the gut, antalarmin could have directly exerted
its effects via a multiplicity of mechanisms. CRH-containing cells have been identified in the human, monkey, cat, and rat stomach and small intestine.70,71 Local
production of CRH has been detected in normal human
colon enterochromaffin cells.72 In patients with ulcerative colitis, considerably enhanced expression of
immunoreactive CRH in mucosal inflammatory cells
has been reported. Intense staining with anti-CRH antibody was also shown in mucosal macrophages and
CRH mRNA was found to be expressed in mucosal
epithelial cells.73
Binding and pharmacological studies have
documented the presence of CRH binding sites in the
circular smooth muscles of the guinea pig cecum.74
CRH binding sites have also been detected in the
myenteric neurons from the guinea-pig ileum,74,75
where it evokes prolonged depolarization in the
majority of these neurons in association with augmented excitability.75 In isolated distal rat colon preparations, CRH provokes a concentration-dependent
increase of both mechanical and electrical peristaltic
activity.76 Urocortin belongs to the CRH family of peptides and CRH-R1 is primarily involved in mediating
479
Figure 2 (a) Effects of normal saline (NT; 0.5 ml), antalarmin
(ANT; 20 mg kg−1 IP), diazepam (DZP; 5 mg kg−1 IM), buspirone (BSP; 4 mg kg−1), fluoxetine (FLX; 10 mg kg−1) and bupropion (BPR; 30 mg kg−1) injected 24 and 2 h before the experiment on immobilization stress-induced gastric ulceration. As
shown, stress ulceration was reduced to ⬍ 9% by antalarmin,
11.5% by diazepam, 28% by bupropion and 47% by fluoxetine. (b) The interior of the rats’ stomachs after the same treatments. Animals groups (n = 6–12) received the same dose of
their drug intraperitoneally twice at −24 h and −2 h of
exposure to immobilization stress for 4 h followed by
immediate termination and collection of trunk blood and stomachs. The latter were opened along the greater curvature
and the sum of each stomach ulcer length (cumulative ulcer
length) was measured with the aid of a magnifying lens and
a micrometer. *P ⬍ 0.05, **P ⬍ 0.01. There was essentially
no ulceration in freely moving animals whether or not they
received any medication.
Molecular Psychiatry
Marked suppression of gastric ulcerogenesis
KE Gabry et al
480
both CRH and urocortin-induced colonic motor
response.43 The expression of the urocortin gene in the
gastrointestinal tract has also been investigated using
reverse transcription polymerase chain reaction (RTPCR) and in situ hybridization histochemistry. PCR has
demonstrated the presence of urocortin mRNA in the
rat brain, duodenum, small intestine, and colon. By in
situ hybridization, urocortin-containing cells are
exclusively localized to the submucosal plexus and
myenteric plexus in the duodenum, small intestine and
Figure 3 Effects of normal saline (NT; 0.5 ml), antalarmin
(ANT; 20 mg kg−1 IP), diazepam (DZP; 5 mg kg−1 IM), buspirone (BSP; 4 mg kg−1), fluoxetine (FLX; 10 mg kg−1) and bupropion (BPR; 30 mg kg−1) on immobilization stress-induced
colonic motility as indicated by the stool pellet output (panel
a) and colonic mucosal mucin content (panel b) in adult male
Sprague–Dawley rats. Colon motility was decreased to 48%
and 49% by diazepam and antalarmin, respectively. Colon
mucin content was increased more than two-fold by antalarmin treatment. *P ⬍ 0.05. In freely moving animals, none of
the drugs significantly caused any significant change in gastric ulceration, colonic mucin or defecation scores as compared to saline treatment.
Figure 4 Effects of normal saline (NT; 0.5 ml), antalarmin
(ANT; 20 mg kg−1 IP), diazepam (DZP; 5 mg kg−1 IM), buspirone (BSP; 4 mg kg−1), fluoxetine (FLX; 10 mg kg−1) and bupropion (BPR; 30 mg kg−1) on struggling scores (panel a), plasma
epinephrine (panel b) and norepinephrine (panel c) after
exposure of adult male Sprague–Dawley rats to 4 h of immobilization stress. All drugs were injected in two full doses at
−24 and −2 h of exposure to immobilization stress in the peritoneal cavity except for diazepam that was administered
intramuscularly. Assays utilized trunk blood samples collected immediately after releasing the animals from the
restrainers. *P ⬍ 0.05, **P ⬍ 0.01. Exposure to this stressor
elevated plasma epinephrine (from 8.40 ± 2.22 to 21.11 ± 2.55
ng ml−1, t = 3.76, P ⬍ 0.01 ) and increased plasma norepinephrine (from 5.45 ± 1.278 to 10.23 ± 0.849 ng ml−1, t = 3.243,
P ⬍ 0.01) as compared to freely moving animals. There was
naturally no struggling behavior in freely moving subjects.
Molecular Psychiatry
Marked suppression of gastric ulcerogenesis
KE Gabry et al
colon.77 More recently, urocortin and CRH-R1 mRNA have
been detected in the human colonic mucosal as well.78
Several lines of evidence have emerged to indicate
bidirectional communication between brain and
enteric CRH systems. CRH pathways have been traced
between the rectum and the brainstem autonomic
nuclei.79 A major symptom in IBS is the exaggerated
pain sensitivity to colon distension, which is correlated with psychological stress.80 CRH released in the
gastrointestinal mucosa from immune cells or enterochromaffin cells may play a role in the modulation of
rectal afferent function as it decreases the thresholds
and increases the intensity for the sensation of discomfort in response to distension. These findings are
consistent with a dual effect of CRH on afferent pathways mediating perception of aversive rectal sensations in humans.81 CRH also mediates the increase in
locus coeruleus spontaneous discharge during colonic
distension in rats.82 Activation of the locus coeruleusnoradrenergic system during colon distension has been
Figure 5 Effect of intraperitoneal CRH (5 ␮g kg−1) on adult
male Sprague–Dawley rats’ colon motility (as indicated by the
stool pellet output: SPO) and mucin (indicated by the number
of mucin-positive colonocytes %) in freely moving animals
4 h after the injections. *P ⬍ 0.05. The effects of intraperitoneal CRH were abolished by antalarmin pretreatment, but
were not affected by vagotomy or sham-vagotomy. *P ⬍ 0.05
when compared to the CRH group.
suggested to serve as a cognitive limb of the peripheral
parasympathetic response and to play a role in disorders characterized by comorbidity of intestinal and
psychiatric symptoms, such as irritable bowel syndrome.82
On the efferent side, CRH acts in the locus coeruleus
to induce a long lasting stimulation of colonic transit
and bowel discharge.83 This mechanism of action may
explain the known clinical efficacy of antidepressants
in IBS. In this study only diazepam was effective in
suppressing colonic responses to stress, perhaps via a
suppressive effect on CRH secretion. By virtue of their
actions on the GABA/benzodiazepine/chloride ionophore complex, benzodiazepines are potent inhibitors
of CRH secretion.84 Our findings here, however, should
not be taken as indices of the efficacy of the drugs
tested, because of their different doses and pharmacokinetic profiles. We have not tested our drugs after multiple administrations to ensure their reaching of the
steady state, which is beyond the scope of this study.
Thus failure of a drug to attenuate a given stressinduced endpoint does not exclude its potential efficacy if administered differently.
Even though antalarmin significantly attenuated
adreno-medullary responses to stress as indicated by
the reductions of plasma epinephrine and norepi-
481
Figure 6 Comparison of the effect of vagotomy vs sham vagotomy (sham.) on stress-induced gastric mucosal lesions in
fasted adult male Sprague–Dawley rats. Vagotomized animals
expressed significantly lower SU scores in response to the
same stressor. CRH administration had no significant effect
on SU in either sham-operated or vagotomized subjects. On
the other hand, antalarmin (Ant) pretreatment further attenuated SU in the sham-operated animals. Means ± SE, *P ⬍ 0.05
compared to the sham-operated group.
Molecular Psychiatry
Marked suppression of gastric ulcerogenesis
KE Gabry et al
482
nephrine, no alteration of catecholamine secretion was
found in response to antidepressants that also attenuated stress ulceration. This supports our previous
notion that suppression of sympathoadrenal responses
to stress may not account for the anti-ulcer effects of
drugs, because sympathectomy actually enhances the
ulcerogenic processes in response to stress.55 Other
studies have shown that neither adrenalectomy nor
hypophysectomy prevent the response of the intestine
to stress, suggesting that neither adrenal nor pituitaryderived factors are responsible for mediating the effects
of stress on the gut.4 Therefore, the weight of evidence
supports the implication of primarily central mechanisms in the pathogenesis of stress ulceration, and largely peripheral mechanisms, presumably utilizing
CRH-R1 in the gut, in the colonic responses to stress.
Taken together, we conclude that brain CRH-R1
exerts an important role in transforming psychological
adversity into visceral damage at least in the stomach.
Our data also indicate that gut CRH-R1 mediates
colonic responses to stress. Novel nonpeptide CRH-R1
antagonists may therefore provide an optimal treatment for such gastrointestinal disorders associated
with stress as stress ulcer and possibly other pathogenetically related conditions such as peptic ulcer and irritable bowel syndrome in humans.
Acknowledgements
We thank Dr Rita J Valentino for her thoughtful discussion and Mr Keith Zachman for his help in conducting the animal experiments.
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