Nakade, et al (R-00499-2004.R1). 1

FINAL ACCEPTED VERSION

Restraint stress delays solid gastric emptying via a central corticotropin-releasing factor and peripheral sympathetic neuron in rats

Yukiomi Nakade, Daisuke Tsuchida, Hiroyuki Fukuda, Masahiro Iwa, Theodore. N. Pappas and Toku Takahashi

Department of Surgery, Duke University Medical Center and Durham Veterans Affairs Medical Center

Durham, NC.

Running title: Stress delays gastric emptying via CRF and sympathetic neuron

Abbreviations: BBB; blood brain barrier, CNS; central nervous system, CRF; corticotropin releasing factor, GI; gastrointestinal, IC; intracisternally, ICV; intracerebroventricularlly, IML; intermediolateral cell column, IP; intraperitoneally, IV; intravenously,

NTS; nucleus tractus solitarius, PVN; paraventricular nucleus, RVLM; rostral ventrolateral medulla, TRH; thyroid releasing hormone

Acknowledgement: This study was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases (RO1 DK55808, T.T.).

Correspondence:Toku Takahashi, MD, PhDSurgical Service 112, VA Medical Center508 Fulton Street, Durham, NC 27705.Phone: 919-286-0411 (Ext.7883)Fax: 919-286-1140E-mail: ttakahas@duke.edu

Articles in PresS. Am J Physiol Regul Integr Comp Physiol (September 30, 2004). doi:10.1152/ajpregu.00499.2004

Copyright © 2004 by the American Physiological Society.

Nakade, et al (R-00499-2004.R1). 2

Abstract

Central corticotropin-releasing factor (CRF) delays gastric emptying through the autonomic

nervous system. CRF plays an important role in mediating delayed gastric emptying

induced by stress. However, it is not clear whether a sympathetic or parasympathetic

pathway is involved in the mechanism of central CRF-induced inhibition of solid gastric

emptying. The purpose of this study was to investigate whether (a): CRF inhibits solid

gastric emptying via a peripheral sympathetic pathway and (b): stress-induced inhibition of

solid gastric emptying is mediated via a central CRF and peripheral sympathetic pathways.

Using male Sprague-Dawley rats, CRF was injected intracisternally with or without various

adrenergic blocking agents. To investigate whether central CRF induced-inhibition of solid

gastric emptying is mediated via a peripheral sympathetic pathway, rats underwent celiac

ganglionectomy one week prior to the gastric emptying study. Ninety minutes after solid

meal ingestion, gastric emptying was calculated. To investigate the role of endogenous CRF

in stress-induced delayed gastric emptying, a CRF type2 receptor antagonist, astressin2-B,

was intracisternally (IC) administered. Rats were subjected to a restraint stress immediately

after the feeding. IC injection of CRF (0.1-1.0 µg) dose-dependently inhibited solid gastric

emptying. The inhibitory effect of CRF on solid gastric emptying was significantly blocked

by guanethidine, propranolol and celiac ganglionectomy, but not by phentolamine. Restraint

stress significantly delayed solid gastric emptying, which was improved by astressin2-B,

guanethidine and celiac ganglionectomy. Our research suggests that restraint stress inhibits

solid gastric emptying via a central CRF type2 receptor and peripheral sympathetic neural

pathway in rats.

Key Words: Blood brain barrier, guanethidine, celiac ganglionectomy

Nakade, et al (R-00499-2004.R1). 3

Introduction

Corticotropin-releasing factor (CRF), one of the stress related neuropeptides, is known to

act in the brain to influence on the gastrointestinal (GI) tract. Exogenously applied CRF into

the central nervous system (CNS) inhibits gastric emptying and acid secretion, while it

stimulates colonic transit through the autonomic nervous system (5, 10, 24, 41). Restraint

stress is known to increase CRF mRNA in the amygdala and paraventricular nucleus (PVN)

(18, 21), resulting in alteration of GI motor activities. Restraint stress-induced alterations of

GI motility are abolished by the central administration of CRF antagonist (26). These data

suggest that endogenous CRF plays an important role in mediating stress-induced

abnormalities of GI motility.

Intracerebroventricularly (ICV) injection of CRF increases plasma insulin (37) and

somatostatin levels (39) and accelerates colonic transit (24) in rats, which are abolished by

vagotomy and atropine. It is suggested, therefore, that the stimulatory effects of central CRF

on GI function are mediated by vagal pathway.

It remains controversial whether the inhibitory effect of central CRF on gastric

emptying is mediated via a parasympathetic (4, 41) or sympathetic pathway (24). Tache et

al. reported that intracisternally (IC)-applied CRF inhibits liquid gastric emptying and that

truncal vagotomy prevented the inhibitory effect of CRF in rats (41). Other investigators

have also shown a role of vagal pathway in the central inhibition of liquid gastric emptying

induced by CRF in rats (4). On the other hand, Lenz et al. reported that sympathetic

blockade, but not vagotomy, prevented the inhibitory effect of CRF on liquid gastric

emptying in rats (24).

In the hepatobilially system, the autonomic nervous system influences hepatic metabolism

and hemodynamics (12). Certain physiological stressors or continuous stimulation of

Nakade, et al (R-00499-2004.R1). 4

sympathetic nerve aggravate hepatic injury (11, 16). For example, central administration of

CRF aggravated carbon tetrachloride acute liver injury through a sympathetic pathway in

rats (44). Acoustic stress inhibits gallbladder contraction via activation of central CRF and

sympathetic efferent in dogs (25). These results suggest that release of norepinephrine,

mediated by central CRF, is the common pathway producing hepatic injury and inhibition of

gallbladder contraction during stress.

There are two distinct CRF receptors, which are coupled to G protein, subtype1 (CRF

type1) and subtype2 (CRF type2). These have been cloned from distinct genes in rodents and

humans (36). CRF type1 receptor activation is associated with the endocrine, behavioral and

autonomic responses to stress (15, 40). In contrast, the activation of the CRF type2 receptor

by CRF agonist in the brain stem imitated stress-induced inhibition of gastric emptying (7,

30). IC-administration of CRF or CRF related peptide, sauvagine and urotensin I, inhibited

solid gastric emptying (29). Vagally stimulated-solid gastric emptying is delayed by

IC-administration of CRF related peptide, urocortin (7). The selective CRF type2 receptor

antagonist, astressin2-B dose-dependently prevented urocortin action while the CRF type1

receptor antagonist, NBI-27914, did not (7).

It is generally accepted that gastric emptying of a liquid meal primarily reflects the activity

of fundus (33). In contrast, gastric emptying of a solid meal is regulated by the coordination

of the antrum, pylorus and duodenum (14, 20, 33). It remains unknown whether a

parasympathetic or sympathetic pathway is involved in mediating central CRF-induced

inhibition of solid gastric emptying.

The present study examined whether a sympathetic nervous pathway is involved in

central CRF-induced inhibition of solid gastric emptying in rats, using combined

pharmacologic and surgical approaches. We also examined whether endogenous CRF is

Nakade, et al (R-00499-2004.R1). 5involved in mediating stress-induced inhibition of solid gastric emptying, using CRF type2

receptor antagonist astressin2-B.

Materials and Methods

Animals

Male Sprague-Dawley rats weighing 240-280 g were housed in-group cages under

conditions of controlled temperature (22-24 oC) and illumination (12 hour light cycle

starting at 6:00 am) for at least seven days before experiments and maintained on laboratory

chow and water. Before the experiment, rats were fasted for 24 hours but given free access

to water. Protocols describing the use of rats were approved by the Institutional Animal

Care and Use Committee of Durham Veterans Affairs Medical Center and in accordance

with the National Institute of Health "Guide for the Care and Use of Laboratory Animals".

Substances and Treatments

A rat/human CRF and astressin2-B (Sigma, St. Louis, MO) were kept in powder form at

–70oC. Peptides were dissolved in 0.9% saline (CRF) or in double-distilled water

(astressin2-B) immediately before use. Under a light anesthesia of isoflurane (3%), rats were

placed in a stereotaxic apparatus (Kopf Model 900, David Kopf Instruments, Tujunga, CA).

Peptides or vehicles were injected IC (10 µl/rat) by puncture of the occipital membrane with

a needle of 10-µl Hamilton microsyringe (Hamilton, Reno, NV). The presence of

cerebrospinal fluid in the Hamilton syringe on aspiration before injection certified the

accuracy of needle placement into the cisterna magna, as previously described (34).

Experimental Design

Measurement of solid gastric emptying

Rats were anesthetized with isoflurane (3%) and mounted on a stereotaxic apparatus. Thirty

minutes after an IC-injection of CRF (0.1-2.0 µg/rat) or saline, preweighed pellets (1.5g)

were given for ten minutes, as previously reported (19, 20).

Nakade, et al (R-00499-2004.R1). 6

Previous report has shown that IC-injection of high dose of CRF (5.0 µg/rat) reduced

food intake from 7 g/ 2 hours to 1 g/ 2 hours (23). If rats did not eat all of 1.5 g meal within

10 minutes, these rats were excluded from the experiment. There were no significant

differences in the time of whole food intake between saline-injected rats (5.2 ± 1.3 min, n=

7) and CRF-injected rats (7.3 ± 2.1 min, n= 7). Ninety minutes after the end of feeding, rats

were sacrificed by the intraperitoneally (IP) injection of pentobarbital (200 mg/kg). The

stomach was surgical isolated and removed. The gastric content was recovered from the

stomach, dried, and weighed. Solid gastric emptying was calculated according to the

following formula, as previously described (19, 20).

Gastric emptying (%) = [1 – (dried weight of food recovered from stomach/weight

of food intake)] ዊ� 100.

Pharmacological and surgical approaches

In order to investigate whether the inhibitory effect of CRF on solid gastric emptying is

mediated through a sympathetic pathway, guanethidine (5.0 mg/kg), phentolamine (1.0

mg/kg) and propranolol (1.0 mg/kg) were injected IP 30 minutes before an IC-injection of

CRF. We have previously proven that an IP-injection of these adrenergic blocking agents

had no significant effects on gastric motility (42) and GI transit (43) in normal conditions in

rats.

To investigate whether inhibitory effect of CRF on solid gastric emptying is mediated

via a parasympathetic or sympathetic pathway, celiac ganglionectomy and truncal vagotomy

were performed one week before the peptide injection. Under pentobarbital (45 mg/kg; IP)

anesthesia, truncal vagotomy was performed by cutting the vagal trunks around the

abdominal esophagus, as previously described (35). A celiac ganglionectomy was carried

out by deflecting the stomach and spleen to the right of the rat, which allowed identification

of the nerves and celiac ganglion (35). Sham operated rats served as controls.

Restraint stress

Nakade, et al (R-00499-2004.R1). 7

After 24 hours fasting, rats were anesthetized with isoflurane and mounted on a stereotaxic

apparatus for an IC injection of astressin2-B (10 µg) or saline (10 µl). We chose the dose of

astressin2-B by the previous reports demonstrating that IC-injection of astressin2-B (10 µg)

prevented restraint stress-induced inhibition of solid gastric emptying (7). Thirty minutes

after the astressin2-B injection, the rats were given a preweighed solid pellet (1.5 g) for ten

minutes. Immediately after the end of feeding, the rats were subjected to the restraint stress.

The rats were restrained in hemicylindrical, well ventilated, Plexiglass tubes for 90

minutes. After the restraint stress loading, the rats were sacrificed by pentobarbital

anesthesia (200 mg/kg, IP). The gastric content was recovered and the solid gastric

emptying was calculated.

To investigate whether the sympathetic pathway is involved in restraint stress-induced

inhibition of solid gastric emptying, we performed guanethidine pretreatment and celiac

ganglionectomy. Guanethidine (5.0 mg/kg) was administered (IP) 30 minutes before an

IC-injection of astressin2-B (10 µg) or saline (10 µl). Celiac ganglionectomy was performed

one week before the restraint stress loading.

To investigate whether peripheral administration of astressin2-B (10 µg) affects the

stress-induced inhibition of solid gastric emptying, astressin2-B (10 µg) was subcutaneously

injected 30 minutes before the feeding.

Statistical Analysis

All results were expressed as means ± SE. The comparison of the values from, between

before and after the restraint stress was calculated by paired Student's t test. A multiple

group comparison was performed by analysis of variance (ANOVA) followed by Scheffe’s

test. A P value < 0.05 was considered statistically significant.

Results

Nakade, et al (R-00499-2004.R1). 8

Effect of sympathetic nerve blockade on the inhibitory effects of CRF on solid gastric

emptying

In saline-treated rats, solid gastric emptying was 62 ± 4% (n=6) 90 minutes after the feeding

of 1.5 g of rat chow. IC-administration of CRF (0.1-1.0 µg) dose dependently inhibited solid

gastric emptying (Fig. 1). Administration of 2.0 µg of CRF did not further inhibit solid

gastric emptying, indicating that the maximal effective dose of CRF on solid gastric

emptying is 1 µg. IC-administration of CRF (1 µg) significantly reduced solid gastric

emptying to 25 ± 3% (n=6).

Pretreatment with noradrenergic blocking agents, guanethidine, partially attenuated

CRF-induced inhibition of gastric emptying to 42 ± 4 % (n=6) (Fig. 2a). Similarly,

pretreatment with b��� adrenoceptor antagonists, propranolol, partially attenuated central

CRF-induced inhibition of gastric emptying to 50 ± 6 % (n=6) (Fig. 2b). In contrast,

pretreatment with alpha adrenoceptor antagonists, phentolamine, did not affect the central

CRF-induced inhibition of gastric emptying (Fig 2b). Celiac ganglionectomy itself had no

significant effects on solid gastric emptying (67 ± 7%, n=5), compared to rats undergoing

sham ganglionectomy (65 ± 6%, n=5). Celiac ganglionectomy completely eliminated the

inhibitory effect of CRF on solid gastric emptying (Fig. 2c).

Truncal vagotomy itself almost completely abolished gastric emptying of solid food (3

± 3%, n=4), compared to rats that underwent sham vagotomy (62 ± 5%, n=5). Therefore, we

were unable to study whether central CRF has the inhibitory effect of on solid gastric

emptying in vagotomized rats.

Effect of intracisternal or subcutaneous injection of astressin2-B on restraint stress-induced

inhibitory effects of solid gastric emptying

Partial body restraint stress reduced solid gastric emptying to 30 ± 3% (n=6). IC-injection

of astressin2-B (10 µg) by itself did not affect solid gastric emptying in the non-restraint rats.

In contrast, IC-injection of astressin2-B (10 µg) almost completely abolished restraint

stress-induced inhibition of solid gastric emptying (Fig. 3a). Subcutaneous injection of

Nakade, et al (R-00499-2004.R1). 9

astressin2-B (10 µg) did not affect restraint stress-induced inhibition of solid gastric

emptying (32 ± 3%, n=5)

Effect of sympathetic nerve blockade on restraint stress-induced inhibitory effects of solid

gastric emptying

Guanethidine restored restraint stress-induced inhibition of solid gastric emptying to 44 ±

1% (n=6) (Fig. 3b). IC-injection of astressin2-B did not show any further improvement of

solid gastric emptying in guanethidine-treated groups (Fig. 3b). Restraint stress delayed

gastric emptying to 35 ± 5% (n=5) in the rats that underwent sham ganglionectomy. Celiac

ganglionectomy almost completely abolished the restraint stress-induced inhibition of

gastric emptying (Fig. 3c).

Discussion

Martinez and colleagues reported that IC-injection of CRF (0.1-1.0 µg) dose dependently

inhibited solid gastric emptying (29). We also demonstrated that IC-injection of CRF

(0.1-1.0 µg/rat) inhibited solid gastric emptying in a dose dependent manner.

Our pharmacological approaches demonstrated that the central CRF-induced inhibition

of solid gastric emptying was significantly restored by the treatment with guanethidine. This

suggests that the adrenergic pathway is involved in mediating CRF-induced inhibition of

solid gastric emptying. This is consistent with the previous study of Lenz. et al. who

reported that delayed gastric emptying induced by central CRF was blocked by adrenergic

blocking agents in rats (24). Adrenoceptors are located in the brain as well as the myenteric

plexus (9). It remains unclear whether CRF-induced inhibition of gastric emptying is via the

peripheral or central adrenergic pathway. Although guanethidine does not cross the blood

brain barrier (BBB) (6), it may act on the area postrema of the brain stem where is loose of

BBB structure.

The present study revealed that celiac ganglionectomy almost completely abolished the

inhibitory effects of CRF on solid gastric emptying. We further demonstrated that central

Nakade, et al (R-00499-2004.R1). 10

CRF-induced inhibition of solid gastric emptying was antagonized by propranolol, not

phentolamine. The activation of beta-adrenoceptors located in the smooth muscle cells

induces cyclic AMP formation, resulting in muscular relaxation of the stomach (3, 42).

These results suggest that central CRF inhibits solid gastric emptying via a sympathetic

pathway and b���-adrenoceptors in conscious rats.

On the other hand, others suggested the involvement of vagal pathways in mediating central

CRF-induced inhibition of gastric emptying. IC-injection of CRF-induced inhibition of

liquid gastric emptying is abolished by vagotomy (41). IC-injection of CRF (21, 63, and 126

pmol) decreases gastric vagal efferent activity in a dose dependent manner (22). This further

supports that CRF may inhibit vagal activity resulting in delayed gastric emptying.

Our current findings suggest that central CRF-induced inhibition of gastric emptying

is mediated via a sympathetic pathway. The discrepancy may be explained by the difference

between liquid and solid gastric emptying.

In liquid meal studies, vagotomy itself did not significantly affect gastric emptying in

rats (41). In contrast, we showed that vagotomy itself significantly delayed solid gastric

emptying. Liquid gastric emptying is believed to be primarily a function of the pressure

gradient between the stomach and duodenum (20, 33). In contrast, gastric emptying of a

solid meal is regulated by the coordination of the antrum, pylorus and duodenum

(antro-pyloro-duodenal coordination) (14, 20, 33). We have previously shown that

vagotomy abolished the postprandial antro-pyloric coordination induced by solid food

ingestion in rats (20). Thus, the vagal nerve may play an important role in regulating solid

gastric emptying.

It has been demonstrated that electrical stimulation of gastric sympathetic nerves

inhibits the overflow of acetylcholine from a vascularly perfused rat stomach (45). It is

conceivable that central CRF activates a sympathetic pathway through which in turn

attenuates vagal cholinergic transmission. This may cause impaired postprandial

antro-pyloric coordination and delayed gastric emptying of solid food.

Nakade, et al (R-00499-2004.R1). 11

Even in liquid emptying study, it is still controversial whether the inhibitory effect of central

CRF on gastric emptying is mediated via a vagal (41) or a sympathetic pathway (24). Tache

et al. used IC-injection of CRF (210 pmol) to induce 81% inhibition of liquid gastric

emptying (41), while Lenz et al. used ICV-injection of CRF (1 nmol) to induce 31%

inhibition of liquid gastric emptying (24). Therefore, it seems that lower dose of

IC-injection of CRF (210 pmol) is more effective than higher dose of ICV-injection of CRF

(1 nmol) to inhibit liquid emptying. As the responsive site of central CRF seems to be a

brain stem (1), it is conceivable that IC-injection of CRF is more potent to inhibit liquid

gastric emptying than ICV-injection. The discrepancy mentioned above may be explained

by the different procedure of CRF injection (ICV vs IC).

Two CRF receptor subtypes (type1 and type2 receptors) have recently been identified

through molecular cloning from distinct genes in rat and human brains (27). CRF type1

receptor activation is believed to be associated with the endocrine, behavioral, autonomic

responses to stress (15, 40). The activation of CRF type2 receptor in the brain stem imitated

stress-induced inhibition of gastric emptying (2, 32), which suggests the involvement of

CRF type2 receptor in stress induced-inhibition of gastric emptying.

We showed that IC-injection of astressin2-B (10 µg) significantly improved restraint

stress-induced inhibition of solid gastric emptying. Subcutaneous administration of high

dose of astressin2-B (200 µg/kg) prevented restraint-induced delay in gastric emptying (32).

It has been reported that peripheral CRF type2 ligand is expressed in the stomach (17). The

possible leakage of astressin2-B from the brain to the periphery may interfere with

stress-induced inhibition of solid gastric emptying. However, our current study showed that

peripheral administration of astressin2-B (10 µg) did not affect restraint stress-induced

inhibition of solid gastric emptying. This indicates that IC-administration of astressin2-B (10

µg) does not affect stress-induced inhibition of solid gastric emptying via the peripheral

circulation.

Nakade, et al (R-00499-2004.R1). 12

Our current study showed that that guanethidine-pretreatment antagonized stress-induced

inhibition of solid gastric emptying. Similarly, celiac ganglionectomy antagonized

stress-induced inhibition of solid gastric emptying. These results strongly suggest the

peripheral sympathetic system is involved in the mechanism of stress-induced inhibition of

solid gastric emptying. IC-injection of astressin2-B did not induce any further improvement

in guanethidine-treated rats. This suggests that stress-induced inhibition of solid gastric

emptying is mediated via central CRF type2 receptors.

The responsible neural pathway mediating activation of sympathetic neurons induced by

central CRF remains to be investigated. CRF receptors and nerve fibers are widely

distributed in CNS (8), concretely located in the nuclei in the medulla and PVN, which are

the important site of sympathetic nervous outflow (1). CRF type2 receptors are located in

nucleus tractus solitarius (NTS) (1).

Neurons within the NTS project to a number of brain stem areas thought to be

involved in the regulation of sympathetic activity (13). NTS also projects efferent nerve to

rostral ventrolateral medulla (RVLM) (38). RVLM is involved in the regulation of

autonomic nervous system, sending projections to the sympathetic intermediolateral cell

column (IML) of the thoracolumbar spinal cord (28). Noradrenergic nerve fibers originating

from cell bodies found in the celiac ganglion innervate to the stomach (31). We injected

CRF into the cisterna magna, which is close to the medulla. Therefore, it can be suggested

that the site of action of CRF is the NTS. It is conceivable that central injection of CRF may

act on the NTS, resulting in activation of RVLM to enhance sympathetic nerve activity.

In summary, the present study indicates that exogenous CRF injected IC acts on the brain

stem to inhibit solid gastric emptying. Restraint stress inhibits solid gastric emptying via a

central CRF in conscious rats. CRF-and restraint stress-induced inhibition of solid gastric

emptying is antagonized by guanethidine and celiac ganglionectomy. Our findings provide

Nakade, et al (R-00499-2004.R1). 13

the first evidence for a role of the sympathetic nervous systems in the central CRF- and

stress-mediated inhibition of solid gastric emptying.

Nakade, et al (R-00499-2004.R1). 14

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Figure legends

Fig. 1 Dose-related inhibition of solid gastric emptying induced by IC-injection of CRF

(0.1-2.0 µg/rat) in 24 hour-fasted rats. CRF or saline was given 10 minutes before a

solid meal injection. Gastric emptying was measured 90 minutes after solid meal

ingestion (n=5-6, *P < 0.05, **P < 0.01 compared with vehicle treated group).

Fig. 2 Effect of guanethidine (a), phentolamine, propranolol (b) and celiac

ganglionectomy (c) on intracisternal injection of CRF (1 µg/rat)-induced delayed

gastric emptying (a). Guanethidine and propranolol, but not phentolamine,

significantly antagonized the inhibitory effect of CRF on solid gastric emptying

(n=5, **P < 0.01 compared with saline injected group. *P < 0.05 compared with

vehicle treated group).

Celiac ganglionectomy also significantly antagonized the inhibitory effect of

CRF on solid gastric emptying (n=5, **P < 0.01 compared with sham operated

group. *P < 0.05 compared with vehicle treated group).

Fig. 3 Effect of astressin2-B (a), guanethidine (b) and celiac ganglionectomy (c) on

restraint stress-induced inhibition of solid gastric emptying. IC-injection of

selective CRF type2 antagonist, astressin2-B, itself had no effects on solid gastric

emptying in non-stressed rats. IC-injection of astressin2-B abolished restraint

stress-induced delayed solid gastric emptying (a) (n=6, **P< 0.01 compared with

control group).

Guanethidine significantly restored restraint stress-induced inhibition of

solid gastric emptying (b). Astressin2-B did not cause any further improvements on

solid gastric emptying in guanethidine treated rats (n=6, **P < 0.01 compared with

astressin2-B treated group).

Nakade, et al (R-00499-2004.R1). 20

Celiac ganglionectomy abolished restraint stress-induced inhibition

of solid gastric emptying (c) (n=5, **P < 0.01 compared with sham-operated

group).

Nakade, et al (R-00499-2004.R1). 21

Vehicle 0.1 0.25 0.5 1

CRF dose (µg)

gast

ric e

mpt

ying

(%

)

∗∗∗

CRF

Saline

2

0

20

40

60

80

∗∗∗∗

Nakade, et al (R-00499-2004.R1). 22

(a)ga

stric

em

ptyi

ng (

%)

0

20

40

60

80

Vehicle Phentolamine Propranolol

CRF (1 µg)

Saline

∗∗ ∗∗

∗(b)

0

20

40

60

80

shamoperation

Celiacganglionectomy

gast

ric e

mpt

ying

(%

)

∗CRF (1 µg)

Saline

∗∗(c)

Vehicle

gast

ric e

mpt

ying

(%

)∗

0

20

40

60

80

Guanethidine

CRF (1 µg)

Saline

∗∗

Nakade, et al (R-00499-2004.R1). 23

Vehicle Restraint

Astressin2-B (10 µg); ic

gast

ric e

mpt

ying

(%

)

0

20

40

60

80 Saline; ic

∗∗

(a)

0

20

40

60

80

shamoperation

Celiacganglionectomy

gast

ric e

mpt

ying

(%

)

Control

Restraint stress

∗

gast

ric e

mpt

ying

(%

)

Vehicle Guanethidine

Restraint + Saline; ic

Restraint + Astressin2-B (10 µg); ic

0

20

40

60

80

∗