Stress-Induced Gastrointestinal Secretory and Motor Responses in Rats Are Mediated by Endogenous Corticotropin-Releasing Factor

GASTROENTEROLOGY 1988;95:1510--7

Stress-Induced Gastrointestinal Secretory and Motor Responses in Rats Are Mediated by Endogenous CorticotropinReleasing Factor H. JURGEN LENZ, ANDREAS RAEDLER, HEINER GRETEN, WYLIE W. VALE, and JEAN E. RIVIER Neurogastroenterology Laboratory, Department of Medicine, University of Hamburg, Hamburg, Federal Republic of Germany; and The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California

Corticotropin-releasing factor (CRF) has been implicated as a central nervous system mediator of stress. This study examined the effects of CRF and stress on gastric secretory and gastrointestinal motor functions in rats. Partial body restraint as a stressproducing stimulus significantly decreased gastric acid secretion, gastric emptying, and small bowel transit but markedly increased large bowel transit. Corticotropin-releasing factor given cerebroventricularly mimicked the gastrointestinal secretory and motor responses induced by partial body restraint. Cerebroventricular administration of a specific CRF receptor antagonist, a-helical CRF-(9-41), but not of the CRF fragment CRF-(1-20), prevented the gastrointestinal secretory and motor responses elicited either by partial body restraint or by exogenous administration of CRF in a dose-dependent fashion. These results suggest that the gastrointestinal secretory and motor responses in rats produced by stress (partial body restraint) are mediated by the endogenous release of CRF. They also indicate that CRF exerts its central nervous system actions on the gastrointestinal tract by a receptor-mediated event.

C

hanges in mood and behavior as well as exposure to stressful stimuli may result in altered gastrointestinal functions that may be associated with abdominal discomfort (1,2). Studies in human subjects with gastric fistulas demostrated that fear, anger, and stress may alter gastrointestinal functions, resulting in decreased gastric secretion, blood flow, and emptying (3,4). An endogenous mediator regulating and coordinating gastrointestinal secretory and motor responses following stress has not been identified. The 41-amino acid peptide corticotropin-releasing

factor (CRF), characterized from ovine and rat hypothalamic extracts (5,6), is released during stress (7). Exogenous administration of CRF into the central nervous system produces endocrine, autonomic, metabolic, cardiovascular, gastric, and behavioral changes that are observed in response to stress (summarized in Reference 8). The central nervous system effects of CRF on gastrointestinal function are characterized by decreases in gastric acid secretion, gastric emptying, and small bowel transit but by marked increases in large bowel transit (9-15). Recent studies using the CRF antagonist, a-helical CRF-(9-41) (16), suggested that endogenous CRF may be involved in mediating the stress-induced release of adrenocorticotropic hormone (ACTH) (16) and epinephrine (17,18), as well as the stressinduced inhibition of growth hormone (19) and luteinizing hormone secretion (20) and stimulation of colonic transit (15). These observations and the anatomic distribution of CRF in brain regions that control gastric secretory and gastrointestinal motor activity (21) prompted us to test the hypothesis that CRF is an endogenous central nervous system mediator of stress-induced changes in gastric acid secretion and gastrointestinal motility. Using the CRF antagonist a-helical CRF-(9-41) and partial body restraint as a stress-initiating stimulus, we now provide evidence that CRF is an endogenous central nervous system mediator of stress-induced gastric secretory and gastrointestinal motor responses in rats.

Abbreviation used in this paper: CRF, corticotropin-releasing factor. © 1988 by the American Gastroenterological Association 0016-5085/88/$3.50

December 1988

Materials and Methods Male Sprague-Dawley rats (200-250 g) were housed under controlled conditions of temperature, humidity, and illumination in single quarters and were fed a standard rat diet. Two days before surgery and the subsequent days after surgery they were fed a liquid diet. The studies were approved by the local animal welfare committee, the Gesundheitsbehorde, Amt fUr Gesundheitswesen und Veteriniirwesen, Hamburg, F.RG. Surgery was performed under general anesthesia using a mixture of xylazine (6 mg/kg Rompun; Bayer AG, Leverkusen, F.RG.) and ketamine-HCl (50 mg/kg Ketanest; Parke Davis & Co., Berlin, F.RG.). Using a stereotaxic instrument (David Kopf Instruments, Tujunga, Calif.), a 22-gauge stainless steel cannula was implanted so that its tip was inside the right lateral ventricle (22). After each experiment, correct placement of the cannula was verified by injection of methylene blue and brain section. To facilitate intravenous injections and aspirations, chronic catheters were implanted in the right jugular vein and subcutaneously exteriorized to exit at the interscapular region of the animal's neck (23). Gastric secretory studies and gastrointestinal motility studies were performed in two different groups of animals. To examine the effects of peptides on gastric acid secretion, a modified Thomas cannula (inner diameter, 3.1 mm; outer diameter, 5.5 mm) (Karolinska Institute, Stockholm, Sweden) was implanted into the most dependent part of the nonglandular portion of the stomach. The cannula was secured to the gastric wall by a purse-string suture, exteriorized through a left paramidline abdominal incision, and closed with an aluminum cap. Five to seven days after gastric surgery, the animals were fitted with a cerebroventricular cannula and an intravenous catheter. All experiments were performed in 24-h fasted rats and 2-4 days after the second operation. Animals that had either residual food or fecal material in their stomachs were not studied. On the day of the experiment, the animals were placed in buckets measuring 15 L and the intravenous catheter and the gastric cannula were connected by polyethelene tubing to infusion and suction pumps, respectively (B. Braun-Melsungen AG, Melsungen, F.RG.). After a I-h basal period, gastric acid secretion was stimulated by intravenous infusion of pentagastrin (16 /Lg/kg . h Gastrodiagnost; E. Merck, Darmstadt, F.RG.). Gastric juice was aspirated and collected at 15-min intervals. A O.l-ml aliquot was titrated with 0.1 N NaOH to pH 7.0 on an automated titration system (Radiometer, Copenhagen, Denmark) and the I-h total acid outputs were calculated (22,23). In some experiments, the effects of CRF (1 nmol) and stress on basal, nonstimulated acid secretion were examined as well. To study the effects of peptides on gastrointestinal motility, silicone catheters (inner diameter, 0.7 mm; outer diameter, 1.1 mm; for the colon, inner diameter, 0.8 mm; outer diameter, 1.6 mm) were implanted into the gastric fundus, the proximal duodenum, and the proximal colon (11). The catheters were secured to the gastrointestinal wall by a purse-string suture and subcutaneously exteriorized to the interscapular region of the animal's neck,

CRF MEDIATES GASTROINTESTINAL STRESS RESPONSE

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where they were secured by adhesive tape. Five to seven days after surgery, cerebroventricular cannulas or intravenous catheters, or both, were implanted. Two to four days later, experiments were performed in 24-h fasted animals that were freely moving in buckets measuring 15 1. Radioactive intestinal markers (New England Nuclear Research Products, Boston, Mass.) were injected as follows: stomach: [3H]polyethylene glycol (mol wt, 800-1000), 1.25 /LCi in 0.5 ml of 0.15 M NaCl; duodenum: 51Cr, 0.4 /LCi in 0.2 ml of 0.15 M NaCl; colon: 51Cr, 0.4 /LCi in 0.2 ml of xanthum gum (Sigma Chemie GmbH, Deisenhofen, F.RG.). After injection of the markers, the tubings were gently flushed with 0.2 ml of 0.15 M NaCl. Thirty minutes after injection of the intestinal markers, the animals were placed in chambers containing carbon monoxide and decapitated. The stomach was removed and the small and large intestine were excised, with care being taken not to dislocate the markers. The stomachs were placed into 60-ml tubes and 0.15 M NaCl was added to yield a total volume of 20 ml. The stomachs were homogenized and then centrifuged at 6000 g for 15 min. An aliquot of 0.2 ml of the supernatant was added to 5 ml of scintillation liquid (Packard Instant Scintillation Gel; Packard Instrument Co., Inc., Zurich, Switzerland). The standard solution was prepared by diluting 0.2 ml of the tritiated polyethelene glycol (1.25 /LCi) with 20 ml of 0.15 M NaCI, of which 0.2 ml was added to the scintillation fluid. Gastric emptying was calculated as 100 - (disintegrations per minute in sample x 100/disintegrations per minute in standard) (11). The small intestine and the large intestine separated from the cecum were each cut into 10 equal segments and their emissions were determined for 1 min in a y-counter. The movement of the marker was assessed by determining the geometric center as previously described (11,24), small numbers indicating slow transit and large nUhlbers indicating fast transit. To study the effects of stress on gastrointestinal secretory and motor responses, an animal model had to be developed that allowed cerebroventricular, intravenous, and intestinal injections as well as intravenous and gastric aspirations. At the time the pentagastrin infusion was started in gastric secretory studies and at the time the intestinal markers were injected in gastrointestinal motility studies, the rats were subjected to stress (partial body restraint) for 60 min and for 30 min, respectively. Each animal was placed on a wooden plate and its trunk was secured to the plate using three metal arcs. The animal was able to move its limbs and head but not its trunk. Under this partial body restraint and after cerebroventricular administration of CRF (1 nmol) or control, several plasma concentrations were determined: ACTH (ACTH J-125 Radioimmunoassay; DRG-Instruments GmbH, Marburg, F.RG.), cortisol (Cortisol Direkt Radioimmunoassay; DRGInstruments GmbH), epinephrine and norepinephrine using a radioenzymatic method (25) and S-[methyl3H]adenosyl-L-methionine (New England Nuclear Products), and glucose (Glucose Analyzer; Beckman Instruments, Palo Alto, Calif.). Neither exogenous CRF (2 nmol) given cerebroventricularly nor restraint (60 min) produced hemorrhagic or ulcerative lesions of the gastroduodenal mucosa on macroscopic examination.

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The peptides were synthesized by solid-phase methodology as previously described (6,16). Amino acid analysis and analytic high-performance liquid chromatography revealed that the peptides were >95% pure. The following peptides were used: rat CRF; a specific CRF receptor antagonist, a-helical CRF-(9-41); the inactive N-terminal fragment, CRF-(1-20); growth hormone-releasing factor; gonadotropin-releasing hormone; canine gastrin-releasing peptide (26); and thyrotropin-releasing hormone. Thyrotropin-releasing hormone was commercially obtained (Bachem, Inc.; Torrance, Calif.). The peptides or the vehicle (sterile water) as a control were given either cerebroventricularly in 5-pJ volumes over a i-min period or intravenously in O.2-ml volumes as a bolus injection followed by a constant infusion (22,23). In gastric secretory studies, peptides or their controls were given cerebroventricularly at the time the pentagastrin infusion was commenced; in motility studies, they were given 30 min before injection of the intestinal markers. The CRF antagonist, the fragment CRF-(1-20), and the vehicle were given 15 min before injection of active peptides or their controls and 15 min before stress exposure. To further delineate the site of action of CRF, the CRF antagonist (10 nmol) was given cerebroventricularly 15 min before intravenous bolus injection of CRF (1 nmol). The data were subjected to analysis of variance, and differences between treatment groups were assessed by the Neuman-Keuls multiple range test (27). The data are expressed as the mean ± SE, and the results are considered significant if p < 0.05.

Results Cerebroventricular administration of CRF resulted in significant and dose-dependent inhibition of gastric acid secretion, gastric emptying, and small bowel transit, but in stimulation of large bowel transit in freely moving rats (Figure 1). Also, CRF (1 nmol) given centrally significantly ~p < 0.01) increased the number of excreted fecal boli from 1.2 ± 0.3 to 4.5 ± 0.6 per 30 min (n = 8). In contrast to CRF, thyrotropin-releasing hormone (1 nmol) administered cerebroventricularly significantly (p < 0.01) increased gastric acid secretion and emptying and small and large bowel transit by 349% ± 29%, 105% ± 11%, 56% ± 8%, and 245% ± 36%, respectively. Other hypothalamic peptides such as growth hormone-releasing factor, gonadotropin-releasing hormone, and CRF-(1-20), given centrally at 1 nmol per animal, did not significantly alter gastric acid secretion and gastrointestinal transit (data not shown). Corticotropin-releasing factor (1 nmol) given cerebroventricularly also decreased basal acid secretion from 11 ± 3 to 4 ± 2 p,moll15 min measured 60 min after CRF injection (p < 0.05). Cerebroventricular administration of increasing doses of the CRF antagonist 15 min before cerebraventricular administration of CRF (1 nmol) dosedependently reversed the central nervous system

GASTROENTEROLOGY Vol. 95, No.6

GASTRIC AtlO SECRElIO

I 10

~

10

d

SHALL BOWEL TRANSIT

.

S .$

i

100 nmol

tR f - Ant

. lnmol tRf

tRF-II'201

Figure 1. Central nervous system effects of CRF, the CRF antagonist (CRF-Ant), and the N-terminal fragment CRF(1-20) on gastric acid secretion (a), gastric emptying (b), small bowel transit (e), and large bowel transit (d) in freely moving rats. The CRF antagonist, CRF-(1-20), and the vehicle (0) were given cerebroventricularly 15 min before central administration of CRF. xp < 0.05, xXp < 0.01 compared with the appropriate control (0); ~p < 0.01 compared with the CRF antagonist (10 nmol) and with the CRF control (0) (n = 6-8 animals in each group).

actions of CRF on gastric acid secretion, gastric emptying, and small and large intestinal transit (Figure 1). The 10-nmol dose of the CRF antagonist completely abolished the central effects of exogenous CRF. In contrast, the 10-nmol dose of CRF(1-20) did not alter the CRF-induced gastric secretory and gastrointestinal motor responses (Figure 1). Similar to CRF, cerebroventricular administration of canine gastrin-releasing peptide significantly (p < 0.01) decreased gastric acid secretion, gastric emptying, and small bowel transit, but increased large bowel transit in freely moving rats (Table 1). Pretreatment of the animals with the CRF antagonist (10 nmol) given cerebraventricularly did not affect the central nervous system actions of gastrin-releasing peptide on gastrointestinal secretory and motor responses (Table 1). Also, the CRF antagonist by itself did not significantly alter any of the gastrointestinal responses that were measured in this study (Table 1). Two nanomoles of CRF given centrally (Figure 2) elicited gastric secretory and gastrointestinal motor responses that were similar to those elicited by central administration of 1 nmol of CRF (Figure 1). Exposure of the animals to stress (partial body re-

CRF MEDIATES GASTROINTESTINAL STRESS RESPONSE

December 1988

Table 1. Central Effects of the Corticotropin-Releasing Factor Antagonist on Gastrointestinal Responses Induced by Gastrin-Releasing Peptide Vehicle Control Gastric acid 250 ± 21 secretion (f.tmol/h) Gastric emptying 93 ± 8

~200

~ 100 ~

CRF antagonist GRP

Control

GRP

29 ± 5

196 ± 26

26 ± 5

1513

J1 ..

100

GASTRIC EMPTYI G

~ 80

i

60 ± 5

91 ± 6

55 ± 5

5.4 ± 0.6

3.0 ± 0.2

5.6 ± 0.8

2.5 ± 0.2

2.8 ± 0.2

7.4 ± 0.7

3.1 ± 0.3

8.0 ± 0.6

60

(%)

Small bowel transit (GC) Large bowel transit (GC)

u

~ 10

.}ARGE BDwEL TRANSIT

!

CRF, corticotropin-releasing factor. GC, geometric center; GRP, gastrin-releasing peptide. The CRF antagonist (10 nmol) or the vehicle (sterile water) was given cerebroventriculariy 15 min before central administration of GRP (1 nmol) or control (0.15 M NaCl). Gastrin-releasing peptide significantly (p < 0.01) altered the gastrointestinal responses in animals receiving the vehicle and in animals receiving the CRF antagonist. n = 5 animals in each group.

straint) produced decreases in pentagastrin-stimulated gastric acid secretion, gastric emptying, and small bowel transit and produced a marked increase in large bowel transit. These gastrointestinal stress responses were similar to those induced by 1 or 2 nmol of CRF given cerebroventricularly (Figure 2). Pretreatment of the animals with increasing doses of the CRF antagonist given cerebroventricularly reversed the stress-induced gastrointestinal responses in a dose-related fashion (Figure 2). The 10-nmol dose of the CRF antagonist completely abolished the gastrointestinal responses induced by partial body restraint. In contrast, the 10-nmol dose of CRF-(1-20) did not significantly alter the stress-induced gastric secretory and gastrointestinal motor responses (Figure 2). Stress also decreased basal acid secretion

Figure 2. Prevention of the stress-induced gastric secretory and gastrointestinal motor responses by the CRF antagonist (CRF-Ant). The CRF antagonist and CRF-(1-20) were given cerebroventriculariy 15 min before exposure of the animals to stress (partial body restraint). For comparison, the left part of this figure depicts the central nervous system effects of exogenous CRF on gastrointestinal functions. xp < 0.05, x xp < 0.01 compared with the appropriate control (0); "'p < 0.01 compared with the CRF antagonist (10 nmol) (n = 6-8 animals in each group).

from 12 ± 3 (before stress exposure) to 5 ± 1 ~moll15 min (60 min after stress exposure) (p < 0.01). To determine the site of action of exogenously administered or endogenously released CRF, studies were carried out injecting the CRF antagonist (10 nmol as a bolus followed by a 10-nmollh constant infusion) intravenously either before central administration of CRF (1 nmol) or before stress exposure. The CRF antagonist given intravenously abolished neither the gastrointestinal secretory and motor re-

Table 2. Peripheral Effects of the Corticotropin-Releasing Factor Antagonist on Corticotropin-Releasing Factor- and Stress-Induced Gastrointestinal Responses Vehicle Control Gastric acid secretion (f.tmol/h) Gastric emptying (%) Small bowel transit (GC) Large bowel transit (GC)

220 90 5.4 3.1

± ± ± ±

31 5 0.3 0.2

CRF 20 68 4.5 8.2

± ± ± ±

4 4 0.2 0.9

CRF antagonist Stress 41 65 4.3 7.9

± ± ± ±

6 6 0.3 0.7

Control 215 88 5.8 2.6

± ± ± ±

31 4 0.3 0.6

CRF 30 66 4.2 7.7

± ± ± ±

6 4 0.3 0.7

Stress 39 69 4.5 7.2

± ± ± ±

6 6 0.2 0.6

CRF, corticotropin-releasing factor; GC, geometric center. The CRF antagonist (10 nmol) or the vehicle (sterile water) was given intravenously as a bolus injection 15 min before central administration of CRF (1 nmol) and control (0.15M NaCl) or 15 min before stress exposure. After bolus injection, the CRF antagonist was given intravenously as a constant infusion at a rate of 10 nmol/h. Intravenous administration of the CRF antagonist did not significantly alter the gastrointestinal responses induced by CRF or stress. n = 5 animals in each group.

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LENZ ET AL.

Table 3. Central Effects of the Corticotropin-Releasing Factor Antagonist on Gastrointestinal Responses Induced by Peripheral Administration of Corticotropin-Releasing Factor Vehicle

CRF antagonist

Control 230 92 5.0 2.9

Gastric acid secretion (J.Lmollh) Gastric emptying (%) Small bowel transit (GC) Large bowel transit (GC)

± ± ± ±

CRF 91 70 4.5 7.7

31 7 0.2 0.2

Control

± 70 ± 7b ± 0.2 ± 0.8 0

244 94 5.3 3.3

± ± ± ±

CRF 105 71 4.7 7.5

31 9 0.3 0.4

± ± ± ±

100 5b 0.3 0.7 0

CRF, corticotropin-releasing factor; GC, geometric center. The CRF antagonist (10 nmol) or the vehicle (sterile water) was given cerebroventricularly 15 min before intravenous bolus injection of CRF (1 nmol) or control (0.15 M NaCl). n = 5 animals in each group. a p < 0.01; b p < 0.05 compared with control.

sponses after central administration of CRF nor those after stress exposure (Table 2). The effects of the CRF antagonist given intravenously were similar to the effects of 0.15 M NaCI given intravenously in both experiments using exogenous CRF and stress experiments (Table 2). Intravenous administration of CRF (1 nmol) also decreased gastric acid secretion and gastric emptying and increased large bowel transit but did not alter small bowel transit (Table 3). Pretreatment of the animals with the CRF antagonist (10 nmol) given cerebroventricularly did not significantly alter the peripheral actions of CRF (Table 3). The central administration of the vehicle for CRF (sterile water) did not produce significant changes in plasma concentrations of ACTH, cortisol, norepinephrine, epinephrine, or glucose (Table 4). However, both the central administration of CRF (1 nmol) and stress exposure resulted in significant (p < 0.01) increases in plasma concentrations of ACTH, cortisol, norepinephrine, epinephrine, and glucose (Table 4). The changes in these parameters after exogenous CRF and after stress were similar (Table 4).

Discussion Various forms of physical or psychologic stress may alter gastrointestinal functions in humans and in animals (1,3,4,28). Some of these stress conditions may cause, or at least be associated with,

diseases such as peptic ulcer (29,30) or the irritable bowel syndrome (2). The central nervous system mediators that underlie stress-induced changes in gastrointestinal functions have not been defined in detail. The hypothalamic peptide CRF (5,6) was implicated as an endogenous mediator of stressinduced endocrine (16,19,20)' autonomic (17,18)' and behavioral responses (31). Exogenous administration of CRF into the central nervous system produces changes in gastrointestinal functions that are characterized by decreases in gastric acid secretion, gastric emptying, and small bowel transit and by an increase in large bowel transit (9-15). These biologic responses are elicited by changes in autonomic nervous system activity (9-11). Using partial body restraint as the stress-initiating stimulus and the CRF antagonist a-helical CRF-(9-41) (16), we tested the hypothesis that CRF serves as an endogenous central nervous system mediator of stress-induced gastric secretory and gastrointestinal motor responses in rats. Cerebroventricular administration of rat CRF resulted in dose-dependent inhibition of gastric acid secretion, gastric emptying, and small bowel transit, but in stimulation of large bowel transit, confirming previous findings (9-13,15). Pretreatment of the animals with a specific CRF antagonist (16), but not with the N-terminal fragment CRF-(1-20), reversed all of the gastrointestinal responses in a dose-related

Table 4. Effects of Corticotropin-Releasing Factor and Stress on the Endocrine and Autonomic Nervous Systems Control

o min ACTH (pM) Cortisol (J.LM) Norepinephrine (nM) Epinephrine (nM) Glucose (mM)

16 0.48 0.70 0.60 4.8

± ± ± ± ±

3 0.1 0.12 0.10 0.3

CRF 20 min

18 0.60 0.72 0.71 5.2

± ± ± ± ±

3 0.1 0.14 0.12 0.4

o min 18 0.51 0.66 0.74 4.9

± ± ± ± ±

3 0.08 0.14 0.12 0.2

Stress 20 min 240 2.44 2.05 1.18 6.9

± ± ± ± ±

27 0.61 0.45 0.20 0.4

o min 16 0.43 0.78 0.52 4.9

± ± ± ± ±

2 0.08 0.11 0.07 0.4

20 min 288 2.73 2.24 1.33 7.8

:±: 31

± ± ± ±

0.45 0.23 0.16 0.6

ACTH, adrenocorticotropic hormone; CRF, corticotropin-releasing factor. Corticotropin-releasing factor (1 nmol) given cerebroventricularly and stress, but not control (sterile water) given cerebroventricularly, significantly (p < 0.01) increased the plasma concentrations of ACTH, cortisol. norepinephrine, epinephrine, and glucose. The zero time point denotes the time of the cerebroventricular injection or the beginning of the stress exposure. n = 5 animals in each group.

December 1988

fashion. These findings imply that CRF exerts its central nervous system action by a receptor-mediated pathway (16-18). However, they do not elucidate the specific site of action of CRF within the brain. Among the hypothalamic releasing factors, the central nervous system actions of CRF appear to be unique as thyrotropin-releasing hormone increased gastric acid secretion, emptying, small bowel transit, and large bowel transit, and growth hormonereleasing factor and gonadotropin-releasing hormone did not alter any of these gastrointestinal responses. The brain-gut peptide gastrin-releasing peptide (26,32) decreased gastric acid secretion, emptying, and small bowel transit but increased large bowel transit after cerebroventricular administration in this study. Pretreatment of the animals with the CRF antagonist did not prevent the gastrointestinal changes that were observed after cerebroventricular administration of canine gastrin-releasing peptide. This finding indicates that the CRF antagonist given centrally appears to be specific for CRF (16). Furthermore, the CRF antagonist given cerebroventricularly by itself did not significantly alter gastric secretion, emptying, or intestinal transit. This suggests that CRF does not appear to participate in the tonic control of gastric acid secretion and gastrointestinal transit in the rat under normal conditions. To examine the role of CRF in mediating stressinduced changes in gastrointestinal functions, an animal model was developed and validated that allowed cerebroventricular, intravenous, and intestinal injections as well as intravenous and gastric aspirations. Partial body restraint of rats was used as the stress-producing stimulus. This form of stress resulted in significant increases in plasma concentrations of ACTH, cortisol, norepinephrine, epinephrine, and glucose, parameters that are frequently elevated in response to various forms of stress (33). Exogenous administration of CRF into the central nervous system also produced increases in plasma concentrations of ACTH, cortisol, norepinephrine, epinephrine, and glucose in this and in previous studies (5,8,10,15,17,18). Therefore, partial body restraint appeared to be a suitable model to further evaluate the role of endogenous CRF in mediating stress-induced gastrointestinal responses. Partial body restraint of rats (a nonulcerogenic stress model in this study) produced gastric secretory and gastrointestinal motor responses that were similar to those produced by exogenous administration of CRF into the cerebroventricular system. Pretreatment of the animals with increasing doses of the CRF antagonist given celltrally reversed the stress-induced responses in a dose-related fashion. These results confirm previously described effects of

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the CRF antagonist on colonic transit (15) and additionally demonstrate that the CRF antagonist abolishes the inhibitory effects of CRF and stress on gastric acid secretion, gastric emptying, and small bowel transit. The highest dose of the CRF antagonist completely abolished the stress-induced changes in gastrointestinal activity, although the same dose of the fragment CRF-(1-20) did not. These findings imply that CRF is endogenously released by the central nervous system in response to partial body restraint in rats. In studies using exogenous CRF as well as in studies using partial body restraint as the stimulus, the CRF antagonist appeared to be more potent in blocking upper than lower gastrointestinal responses. This may be due to distinct central nervous system sites that control the upper and lower gut, and therefore due to different concentrations of the CRF receptor antagonist at its target cells, or merely due to the more pronounced response of the large intestine after CRF administration and stress exposure. Intravenous administration of the CRF antagonist did not affect the gastrointestinal responses that were elicited either by cerebroventricular administration of CRF or by partial body restraint. These observations imply that both exogenous and endogenous CRF acts within the central nervous system to alter gastrointestinal functions, and does not act by its appearance in the peripheral circulation (34) or by its action on the pituitary gland (11). Intravenous administration of CRF also inhibited gastric acid secretion and emptying and stimulated large bowel transit, confirming previous observations (11-15,35). However, cerebroventricular administration of the CRF antagonist at a dose that was 10-fold greater than that of CRF given intravenously did not abolish the peripheral effects of CRF. These observations further strengthen the contention that CRF administered cerebroventricularly acts within the brain and that CRF administered intravenously acts at an as yet unidentified peripheral site. It is of interest that functional CRF receptors have been identified in the primate peripheral sympathetic nervous system (36) that may serve as a target site for peripheral CRF. The study by Williams et al. (15) showed that intravenous or cerebroventricular administration of the CRF antagonist reversed the stimulatory effect of stress on colonic transit but not the inhibitory effect on small bowel transit. The present investigation demonstrated that cerebroventricular administration of the CRF antagonist resulted in a dose-dependent attenuation of the inhibitory effects produced by stress and CRF on the small intestine, whereas the previous report (15) showed that a single dose of the CRF antagonist was ineffective in preventing the

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response of the small intestine to stress. As our study and that of Williams et al. used different stress models (physical restraint versus wrap restraint), it is possible that different factors (Le., pathways or target sites) are involved in mediating the effects of stress on the small intestine. Differences in the stress models may also explain the discrepant findings that intravenous administration of the CRF antagonist did not abolish the stress-induced colonic response in this report but did in the previous report (15). In the present study, but not in that of Williams et al., cerebroventricular, intravenous, and intestinal injections were performed in freely moving animals and no ether was used before stress exposure. At present, it is unknown how these variables may alter gastrointestinal stress responses. It is unlikely that the dose of the CRF antagonist in this study was insufficient, because the intravenous bolus injection of CRF antagonist (10 nmol) followed by a continuous infusion (10 nmol/h) completely abolished the peripheral effects of 1 nmol of CRF on acid secretion, emptying, and large bowel transit (unpublished observations) . Some forms of stress may be associated with decreased gastric blood flow (37), which renders the gastric mucosa more vulnerable to exogenous noxious agents and to endogenous aggressive factors (such as gastric acid) that playa role in the pathogenesis of peptic ulcer disease (38). Therefore, a decrease in gastric acid secretion mediated by CRF during stress may serve as a protective mechanism to diminish the occurrence of peptic ulcers. Furthermore, CRF produces intestinal motor abnormalities that resemble those of the irritable bowel syndrome, whose symptoms are markedly influenced by psychologic factors and stressful life situations (2). The importance of CRF in gastrointestinal diseases remains to be determined. Finally, different forms of stress in humans (3,28-30) and in animals (1,39,40) may produce different gastrointestinal responses (Le., an increase rather than a decrease in gastric acid secretion) and, therefore, CRF may not be the only central nervous system mediator of stress-induced changes in gastrointestinal function. In summary, the results of this study indicate that the gastrointestinal secretory and motor responses induced by partial body restraint of rats as a stressproducing stimulus are mediated by the endogenous release of CRF. They also suggest that CRF exerts its central nervous system actions on the gastrointestinal tract by a receptor-mediated pathway and that peripheral CRF may alter gastrointestinal function via peripheral, as yet unidentified, target sites. The central nervous system effects of CRF described in this report are compatible with the contention that CRF may serve as an endogenous central nervous

GASTROENTEROLOGY Vol. 95, No.6

system mediator that initiates and coordinates gastrointestinal secretory and motor responses during some forms of stress.

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Received January 25, 1988. Accepted July 8, 1988. Address requests for reprints to: H. J. Lenz, M.D., Department of Medicine, University of California at San Diego Medical Center, 225 Dickinson Street, San Diego, California 92103. This work was supported by grant L2 470/2-1 from the Deutsche Forschungsgemeinschaft, by the Hamburgische Wissenschaftliche Stiftung, and by grant AM26741 from the National Institutes of Health. The authors thank Michael Burlage, Erika Friemel, Karin Feutlinske, Ron Kaiser, and Robert Galyean for technical assistance.