1 CURE: Digestive Diseases
Research Center, Urocortin, a new
mammalian member of the corticotropin-releasing factor (CRF) family has
been proposed to be the endogenous ligand for CRF receptor 2 (CRF-R2).
We studied the influence of intravenous urocortin on gastric emptying
and the role of CRF-R2 in peptide action and postoperative gastric
ileus in conscious rats. The intravenous doses of rat CRF and rat
urocortin producing 50% inhibition of gastric emptying were 2.5 and
1.1 µg/kg, respectively. At these intravenous doses, CRF and
urocortin have their actions fully reversed by the CRF-R1/CRF-R2
antagonist astressin at antagonist/agonist ratios of 5:1 and 67:1,
respectively. Astressin (12 µg/kg iv) completely prevented abdominal
surgery-induced 54% inhibition of gastric emptying 3 h after surgery
while having no effect on basal gastric emptying. The selective
nonpeptide CRF-R1 antagonists antalarmin (20 mg/kg ip) and NBI-27914
(400 µg/kg iv) did not influence intravenous CRF-, urocortin- or
surgery-induced gastric stasis. These results as well as earlier ones
showing that
corticotropin-releasing factor; NBI-27914; antalarmin; astressin; postoperative gastric ileus; abdominal surgery
CORTICOTROPIN-RELEASING factor (CRF) was originally
characterized by Vale et al. (53) from ovine hypothalami in 1981 and named for its property to stimulate anterior pituitary secretion of
ACTH and CRF mediates its actions through activation of specific, seven
transmembrane domain receptors, which are coupled to a guanine nucleotide stimulatory factor
(Gs) signaling protein resulting in increased intracellular cAMP levels (6, 51). To date, two CRF
receptor subtypes, designated CRF-R1 and CRF-R2, were identified
through molecular cloning from distinct genes in the rat and human (6,
24). CRF-R2 exists in multiple forms ( Urocortin, a new 40-amino acid mammalian member of the CRF family, was
cloned recently from rat midbrain and detected at peripheral sites,
including the rat gut and heart and human lymphocytes (1, 10, 54).
Urocortin possesses the characteristics of an endogenous ligand for
CRF-R2 (10, 54). In Chinese hamster ovary cells, which have a stable
expression of CRF-R2 To better define the inhibitory effects of peripheral CRF on gastric
motor function, we studied the influence of the newly characterized
mammalian member of the CRF family, rat urocortin injected
intravenously, on gastric emptying of a nonnutrient liquid meal and
compared its potency with that of rat CRF in conscious rats. Then we
tested the blocking action of the CRF-R1/CRF-R2 antagonist astressin
(37) or the selective CRF-R1 antagonists antalarmin (55) and NBI-27914
(7) administered peripherally against intravenous CRF-, intravenous
urocortin-, and stress-related inhibition of gastric emptying caused by
abdominal surgery.
Animals.
Male Sprague-Dawley rats (Harlan, San Diego, CA), weighing about 250 g
(range 230-280 g), were housed under controlled conditions of
12:12-h light-dark cycle with room temperature maintained at 22 ± 1°C. Animals were allowed free access to food (Purina Rat Chow) and
tap water. Before the experiment, rats were deprived of food for
18-20 h, whereas water was provided ad libitum up to the beginning
of treatment. Experiments were performed under the Veterans Affairs
Animal Component of the Research Protocol number 96-080-08.
Drugs.
Rat/human CRF (r/hCRF), rat urocortin, and astressin,
cyclo-(30-33)-[D-Phe12,Nle21,38,Glu30,Lys33]
r/hCRF12 Measurement of gastric emptying.
Measurement of gastric emptying was performed as previously detailed
(49). The liquid meal consisted of methyl cellulose (Sigma Chemical,
St. Louis, MO) dispersed in hot water at a final concentration of 1.5%
under continuous stirring in which phenol red (50 mg/100 ml, Sigma) was
added as a nonabsorbable marker. The meal (1.5 ml/rat) was given to
conscious rats by oral intubation using stainless steel cannulas, and
20 min later rats were euthanized by
CO2 inhalation. The abdominal
cavity was opened, the gastroesophageal junction and the pylorus were
clamped, and then the stomach was extirpated and rinsed in 0.9%
saline. After the clamps were removed the stomach was placed in 100 ml
of 0.1 N NaOH and homogenized (Polytron, Brinkmann Instruments). The
suspension was allowed to settle for 1 h at room temperature, and 5 ml
of the supernatant were added to 0.5 ml of 20% TCA (wt/vol) and then
centrifuged at 3,000 rpm at 4°C for 20 min. The supernatant was
mixed with 4 ml of 0.5 N NaOH, and the absorbance of the sample read at
560 nm (Shimazu 260 Spectrophotometer). Phenol red recovered from stomachs immediately after the administration of the meal was used as
standard (0% emptying). Percent emptying in the 20-min period was
calculated according to the following equation: percent emptying = (1 Experimental protocols.
The experimental design included vehicle and several doses of test
substances evaluated on the same day.
Effects of intravenous CRF and urocortin on gastric emptying.
Rat CRF, rat urocortin (0.4, 1.2, 2.4, or 4.0 µg/kg in 0.1 ml), or
saline (0.1 ml) was injected intravenously through the jugular vein in
rats under short enflurane anesthesia (5.5% vapor concentration in
oxygen; Ethrane-Anaquest, Madison, WI). The intravenous doses of CRF
were based on previous dose-response studies (49). After intravenous
injection animals were returned to their home cages, 10 min thereafter
the methyl cellulose phenol red meal was administered per orogastric
gavage in lightly restrained rats, and 20 min later animals were
euthanized to measure gastric emptying.
Effects of peripheral CRF antagonists on intravenous CRF- and
urocortin-induced inhibition of gastric emptying.
In rats under short enflurane anesthesia, astressin (4, 12, 40, or
80 µg/kg in 0.1 ml) or its vehicle (0.1 ml distilled water) or NBI-27914 (400 µg/kg in 0.1 ml) or its vehicle (16% DMSO and 84%
PBS in 0.1 ml) was injected intravenously immediately before that of
CRF (2.4 µg/kg in 0.1 ml), urocortin (1.2 or 2.4 µg/kg in 0.1 ml),
or saline (0.1 ml). In other groups, antalarmin (20 mg/kg in 0.3 ml) or
its vehicle (distilled water containing 8% cremophor EL and 8%
ethanol in 0.3 ml) was injected intraperitoneally in conscious rats 1 h
before intravenous injection (0.1 ml) of CRF (2.4 µg/kg), urocortin
(1.2 µg/kg), or saline under enflurane anesthesia. The
intravenous doses of astressin were based on the previous dose-related
antagonism of CRF action on gastric emptying on intracisternal
injection of both peptides (25). Antalarmin was administered under
similar conditions previously reported to block ACTH release induced by
systemic CRF in rats (55) and that of NBI-27914 on in vitro inhibition
of CRF binding in cells stably transfected with human CRF-R1 receptor
(7). After the administration of CRF, urocortin, or saline, rats were
returned to their home cages, 10 min later the liquid meal was
administered, and 20 min later animals were euthanized to measure
gastric emptying.
Effects of CRF antagonists on abdominal surgery-induced inhibition
of gastric emptying.
Under a 10-min exposure to enflurane anesthesia (5.5% vapor
concentration in oxygen), groups of rats were injected intravenously either with astressin (4, 12, or 40 µg/kg, 0.1 ml) or distilled water
(0.1 ml) or NBI-27914 (400 µg/kg in 0.1 ml) or its vehicle (16% DMSO
and 84% PBS in 0.1 ml) immediately before abdominal surgery with cecal
manipulation performed as previously described (3). In other groups
antalarmin (20 mg/kg in 0.3 ml) or its vehicle (distilled water
containing 8% cremophor EL and 8% ethanol in 0.3 ml) was injected
intraperitoneally in conscious rats 1 h before the abdominal surgery
with manipulation of the cecum. Briefly, abdominal surgery consisted of
a medial celiotomy (3-4 cm) and cecal exteriorization and handling
in gauze soaked with saline for a 1-min period, and then the cecum was
returned to the abdominal cavity. The linea alba and the skin were
closed separately with 3-0 silk sutures. Sham-operated control groups were exposed to a similar duration of enflurane anesthesia (10 min) and
had only a skin incision with no laparotomy and manipulation of the
cecum. The phenol red methyl cellulose meal was administered at 160 min
after the end of the surgery and gastric emptying was determined 20 min later.
Statistical analysis.
All results represent means ± SE. For two-group comparisons, data
were analyzed by Student's t-test.
Comparisons between multiple groups were performed using one-way ANOVA
followed by a Student-Newman-Keuls multiple-comparison test.
Differences were considered statistically significant if
P < 0.05. The intravenous doses of
CRF and urocortin inhibiting gastric emptying by 50% were determined
using nonlinear regression to sigmoidal equation with variable slope
(Prism, GraphPad, San Diego, CA) and intravenous saline-treated group
as 0% inhibition.
Effects of intravenous CRF and urocortin on gastric emptying.
In rats injected intravenously with saline, 52.9 ± 1.8%
(n = 20) of the noncaloric methyl
cellulose liquid meal was emptied after 20 min. Rat CRF injected
intravenously (1.2-2.4 µg/kg) inhibited gastric emptying in a
dose-dependent manner [ANOVA,
F(4,37) = 10.393, P < 0.001; Fig.
1]; a plateau inhibitory response was observed at 2.4 and 4.0 µg/kg (24.2 ± 7.9%,
n = 5, and 25.5 ± 4.5%,
n = 6, respectively), whereas at 0.4 µg/kg intravenous CRF did not significantly reduce gastric emptying
(42.8 ± 5.4%, n = 7; Fig.
1).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical CRF9
41
(a CRF-R2 more selective antagonist) partly prevented postoperative
ileus indicate that peripheral CRF-R2 may be primarily involved in
intravenous urocortin-, CRF-, and abdominal surgery-induced gastric stasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-endorphin. CRF in the brain plays a prominent role in
integrating the efferent components of the behavioral, endocrine, autonomic, and immune responses to stress (13, 33, 51). Recent evidence
indicates that peripheral CRF also exerts specific biological actions
that may have physiological relevance in the modulation of behavior,
immune, pain, and visceral responses to stress (16, 30, 44, 50). In
particular, peripheral administration of CRF inhibits gastric emptying
in rats, mice, and dogs (50), and intravenous injection of the CRF
antagonist
-helical CRF9
41 partially prevents postoperative gastric ileus in rats
(4).
and
) as splice variants
differing in their amino acid
NH2-terminal extracellular domains
and distribution (6). In human brain, unlike in rat brain, another
CRF-2
splice variant has been recently identified (18). CRF-R2
is
located on brain neurons, whereas CRF-R2
is found in nonneuronal
brain tissue and in the periphery (predominantly in the heart and the
gastrointestinal tract in rats) (24, 36).
, rat urocortin, and nonmammalian members of the
CRF family, sauvagine and urotensin I exhibit a much higher binding
affinity and stimulation of cAMP than rat CRF, whereas urocortin,
sauvagine, and urotensin I display almost similar binding affinity to
CRF-R1 as to CRF (10, 37, 43, 54). Therefore, the distinct
pharmacological profiles of existing ligands for the CRF-R1 and CRF-R2
have been used to discriminate their localization and involvement in
biological actions of CRF (42, 43). In addition, recent advances
have been made in the development of CRF receptor antagonists.
Astressin, cyclo-(30-33)-[D-Phe12,Nle21,38,Glu30,Lys33]
rat and human (r/h) CRF12
41,
which has a low intrinsic activity and high affinity to both CRF-R1 and
CRF-R2, is more potent than any other peptide CRF antagonists reported
to date to antagonize intravenous CRF action at the pituitary (12, 37). Moreover, nonpeptide CRF antagonists such as NBI-27914, SC-241, CP-154,526, and the CP-154,526-related compound antalarmin display selectivity for CRF-R1 (7, 8, 43, 55), whereas the peptide antagonist
-helical CRF9
41 has a higher
affinity at the CRF-R2 than CRF-R1 (17, 37). Convergent sets of
evidence established that peripheral CRF-induced stimulation of ACTH
secretion at the pituitary level involved CRF-R1 (46, 51), whereas the
relaxing action of CRF on mesenteric artery or anti-inflammatory effect of systemically injected CRF and urocortin appear to be mediated by
CRF-R2 (11, 42, 52).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
41 (Salk Institute,
Clayton Foundation Laboratories for Peptide Biology, La Jolla, CA),
were synthesized and purified as previously described (12). Peptides
were kept in powder form at
70°C and dissolved immediately
before use. CRF and urocortin were dissolved in sterile saline and
astressin in double-distilled water (adjusted to pH 7.0 and warmed to
37°C). NBI-27914 (Neurocrine Biosciences, San Diego, CA) was
synthesized as a tosylate salt as previously described (7). Before use
NBI-27914 was dissolved in 100% DMSO at a concentration of 10 mM and
further diluted with PBS, pH 7.4. Antalarmin was synthesized and
dissolved as previously described (55) using 50% ethanol and 50%
cremophor EL (PEG-35 castor oil) and then further diluted with
distilled water.
absorbance of sample/absorbance of standard) × 100.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Dose-dependent inhibition of gastric emptying of noncaloric liquid meal
induced by intravenous injection of corticotropin-releasing factor
(CRF) or urocortin in conscious rats. Peptide or saline was injected
intravenously under short enflurane anesthesia 10 min before orogastric
administration of meal. Gastric emptying was measured 20 min later.
Each point represents mean ± SE of 4-20 animals/group.
* P < 0.05 vs. intravenous
saline-injected group (ANOVA followed by Student-Newman-Keuls
multiple-comparison test). # P < 0.05 vs. respective CRF group (Student's
t-test).
Effects of intravenous astressin on intravenous CRF- and
urocortin-induced inhibition of gastric emptying.
In rats injected intravenously with water followed by saline (0.1 ml
each), 54.4 ± 2.7% (n = 12) of
the nonnutrient meal was emptied from the stomach 20 min after its
administration. CRF (2.4 µg/kg) or urocortin (2.4 µg/kg)
significantly reduced gastric emptying to 23.9 ± 6.1%
[ANOVA, F(6,40) = 7.22, P < 0.001, n = 6] and 12.7 ± 2.0%
[ANOVA, F(6,47) = 25.5, P < 0.0001, n = 13], respectively,
in intravenous water-pretreated rats (Fig.
2). Astressin (40 or 80 µg/kg iv) had no
significant effect on basal gastric emptying, although at 80 µg/kg
there was a tendency to reduce gastric emptying (Table
1).
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|
Effects of peripheral antalarmin or NBI-27914 on intravenous CRF- or
urocortin-induced inhibition of gastric emptying.
CRF (2.4 µg/kg) or urocortin (1.2 µg/kg) injected intravenously
significantly inhibited gastric emptying in vehicle-pretreated rats
(Table 2). Antalarmin (20 mg/kg ip)
modified neither basal nor intravenous CRF- or urocortin-induced
inhibition of gastric emptying (Table 2). No antagonist-to-agonist
ratio could be deduced due to the different routes of administration.
NBI-27914 (400 µg/kg iv) had no significant effect on the basal
gastric emptying or intravenous CRF- and urocortin-induced gastric
stasis (Table 2). The dose of NBI-27914 tested corresponds to an
antagonist-to-agonist ratio (µg/kg) of 167:1 for CRF and 333:1 for
urocortin.
|
Effects of CRF antagonists on abdominal surgery-induced inhibition
of gastric emptying.
For control rats exposed to 10 min of anesthesia in which an
intravenous injection of water followed by abdominal skin incision without laparotomy were performed, gastric emptying was 59.5 ± 2.8% (n = 10) as measured during the
160- to 180-min period after the end of anesthesia. Abdominal surgery
and cecal manipulation in rats injected intravenously with water
inhibited gastric emptying to 27.5 ± 3.2% at the 160- to 180-min
period after surgery [P < 0.05 vs. vehicle plus control, n = 11, ANOVA, F(6,35) = 33.86, P < 0.0001]. Astressin at
intravenous doses of 12 or 40 µg/kg completely antagonized
postoperative gastric ileus (58.0 ± 1.2 and 54.1 ± 4.6%,
respectively, P < 0.05 vs. vehicle
plus surgery, n = 4-5),
whereas it had no influence on basal gastric emptying in sham control
(58.1 ± 3.7 and 53.2 ± 8.0%, respectively,
n = 3-4 for each dose,
P > 0.05 vs. water plus sham
operation; Fig. 3). The CRF-R1 antagonists
antalarmin (20 mg/kg ip) or NBI-27914 (400 µg/kg iv) did not alter
abdominal surgery-induced inhibition of gastric emptying (Table
3).
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DISCUSSION |
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Present and previous studies established that CRF decreases gastric emptying of a nonnutrient liquid meal in rats, mice, and dogs after intravenous (4, 34, 38, 45, 49, 56) as well as intraperitoneal or subcutaneous administration in rats or mice (5, 20, 45). We showed that rat urocortin, a novel mammalian member of the CRF family, injected intravenously also inhibits gastric emptying of a noncaloric liquid meal in rats. Urocortin is structurally related to sucker fish urotensin I (63% sequence identity) (19) and shares 45% sequence identity with r/hCRF and 35% with sauvagine (10, 54). Likewise, urotensin I and sauvagine injected subcutaneously suppress gastric emptying in rats (5, 15). Taken together, these findings indicate that mammalian and nonmammalian homologs of CRF display a similar pattern of action on gastric motor function when injected peripherally. Urocortin inhibits gastric emptying at a similar or lower intravenous dose range (100-500 pmol/kg) than cholecystokinin, peptide YY, and glucagon-like peptide 1 (14, 35, 39). Only a few studies have reported biological actions of peripherally administered urocortin at such a dose range, and they relate to the modulation of pituitary secretion, blood pressure, and inflammation in rats (52, 54). The present data provide the first evidence that peripherally injected urocortin influences gut function.
Defined differences in CRF-R1 and CRF-R2 pharmacology provide a tool with which to distinguish the involvement of the two receptor subtypes through which CRF actions are mediated (6). In particular, urocortin, sauvagine, and urotensin I exhibit much greater affinity than CRF for the CRF-R2 isoforms (10, 37, 54). In the present study, urocortin was found to be 2.3-fold more potent than CRF to inhibit gastric emptying. Because both peptides used were derived from the rat sequence, possible differences related to nonhomologous species comparison can be ruled out. A rank order of potency of nonmammalian CRF-related peptides (sauvagine > urotensin I > CRF) to inhibit gastric emptying of a nonnutrient liquid meal after subcutaneous injection in rats was also reported (15, 25). Compared with CRF, the greater potency of urocortin, urotensin I, and sauvagine injected peripherally to delay gastric emptying is in keeping with their relatively higher affinities at the CRF-R2 (10, 43, 54).
In addition, the use of CRF receptor antagonists further supports the
speculation that the inhibition of gastric emptying induced by members
of the CRF family may be mediated through CRF-R2. Both antalarmin and
NBI-27914 are selective CRF-R1 antagonists (7, 22, 55) and did not
modify intravenous CRF- or urocortin-induced gastric stasis. Antalarmin
is a derivative of the well-established CRF-R1 antagonist CP-154,526
(8, 55) and was administered under identical conditions to those
inhibiting the CRF-R1-mediated effect of intravenous CRF (4.7 µg/rat)
on pituitary ACTH release (55). NBI-27914 (606 mol wt) was administered
at a 193-fold higher molar dose than astressin. Because both astressin
and NBI-27914 have a similar affinity at the CRF-R1
(Ki in the 2 nM
range) (7, 12, 37), we can assume that the NBI-27914 at the intravenous dose used should be efficient to block peripheral CRF-R1. By contrast, astressin, which exhibits a mixed CRF-R1/CRF-R2 antagonist profile (12,
37), completely abolished intravenous CRF-induced 50% inhibition of
gastric emptying when injected intravenously at a low dose (12 µg/kg). This shows the high potency of the new CRF antagonist as
previously established in vitro and in vivo mainly against intravenous
CRF-induced ACTH release (40). The higher ratio of astressin to
urocortin (67:1) than astressin to CRF (5:1) required to antagonize
intravenous urocortin action is consistent with the greater affinity of
urocortin for CRF-R2 ( and
) compared with CRF (10, 37, 43, 54).
Recent studies indicate that
-helical
CRF9
41 is more selective to
antagonize CRF-R2 based on its much greater affinity at CRF-R2 than
CRF-R1 (37) and its differential antagonistic activity for intravenous CRF actions on the pituitary (antagonist-to-agonist ratio of 3,000:1) vs. cardiovascular system (6:1) (11). We previously showed that
-helical CRF9
41 injected
intravenously prevented intravenous CRF at a much lower ratio (100:1)
than required to block intravenous CRF-induced ACTH release (4, 11).
With the use of rank order of potency of CRF-related peptides and
-helical CRF9
41-to-CRF ratio
as a strategy to discriminate CRF receptor subtypes, as well as the
presence of CRF-R2 in the heart and vessels, the peripheral cardiovascular effects of CRF were suggested to involve CRF-R2 (11, 17,
42), whereas the effect of intravenous CRF to stimulate pituitary ACTH
release was mediated by CRF-R1 (46, 51). Taken together, the present
data provide convergent evidence that CRF-R2 may be preferentially
involved in intravenous CRF-induced delay of gastric emptying and that
such a response is not secondary to pituitary activation because
nonpeptide CRF-R1 antagonists have no effect. However, the role of
CRF-R2 will ultimately be assessed when CRF-R2 antagonists/agonists
devoid of affinity for CRF-R1 are developed.
CRF injected into the cerebrospinal fluid at a similar dose range
inhibits gastric motor function, and CRF-R2 in the brain was suggested
to mediate CRF action (25). However, it is unlikely that the inhibition
of gastric emptying induced by intravenous CRF reflects a central
action due to the entry of CRF across the blood-brain barrier (2).
First, the characteristics of pharmacokinetic studies in mice indicate
that peripheral CRF is not transported from the blood to the brain
(28). Second, astressin (40 µg/kg) injected intravenously did not
alter the 50% inhibition of gastric emptying induced by intracisternal
injection of CRF (26), whereas it prevented the effect of intravenous
CRF (present study). The localization of peripheral CRF-R2 receptors on
which CRF and urocortin act needs to be further assessed. CRF-R2 are
expressed in the gastrointestinal tract (36), although the exact
cellular distribution has not yet been established. In addition, CRF
binding sites have been found on the subdiaphragmatic vagus (31). These
pathways may have relevance as vagotomy attenuated peripheral
CRF-induced delay of gastric emptying in rats (5), whereas blockade of sympathetic nervous system by adrenalectomy, celiac ganglionectomy, or
bretylium had no effect (20, 38). The extrinsic and/or intrinsic neural
pathways vs. direct action of intravenous urocortin on gastric smooth
muscles needs to be further investigated.
Astressin injected intravenously did not influence gastric emptying,
suggesting that peripheral CRF receptors are not involved in the basal
regulation of gastric emptying of a nonnutrient liquid meal. By
contrast, under stress-related conditions, such as abdominal surgery,
known to activate CRF pathways and to increase circulating CRF levels
(32), the activation of peripheral CRF receptors plays a role in the
alterations of gastric motor function. In the present study, laparotomy
followed by 1-min manipulation of the cecum results in 47% reduction
of gastric transit monitored 3 h later in agreement with our previous
studies (3, 4). Intravenous injection of astressin at 12 µg/kg
abolished the abdominal surgery-induced inhibition of gastric emptying
monitored 3 h later, whereas nonpeptide CRF-R1 antagonists have no
effect. In a previous study, -helical
CRF9
41 injected intravenously at
200 µg/kg normalized only by 70% gastric transit 3 h after abdominal
surgery while completely reversing intravenous CRF-induced similar
inhibition of gastric emptying (4). The enhanced potency of astressin compared with
-helical CRF9
41
may be related to the unique properties of astressin which has low
intrinsic activity and binding affinity to the CRF binding protein as
well as a greater affinity at CRF receptors than
-helical
CRF9
41 (12, 37). These results
indicate that peripheral CRF receptors, most likely the CRF-R2 located
in the periphery (23), may play a key role in mediating acute
postoperative gastric ileus.
We previously reported that intracisternal injection of astressin at a
similar dose range also prevented abdominal surgery- and cecal
manipulation-induced gastric ileus assessed 3 h after surgery (27).
These findings would suggest that both central and peripheral CRF
receptors are involved in postoperative gastric ileus (Ref. 3 and
present study). As recent reports demonstrated an active transport of
CRF from the brain to the periphery, where the peptide could directly
affect a peripheral organ (28, 29), a possible dual action of peptide
receptor antagonists needs to be further investigated using
intracisternal injection of astressin and peripheral administration of
CRF. This will allow us to further ascertain central and/or peripheral
sites of astressin action to reverse postoperative gastric ileus
particularly since experiments were performed during a 3-h period,
which allows maximal peptide transport from the brain to the periphery
as shown by kinetic studies (28, 29). By contrast, other acute
stressors (restraint, forced swimming, ether, intravenous
interleukin-1) that induce inhibition of gastric emptying have been
reported to be selectively blocked by central, but not by peripheral,
injection of -helical CRF9
41
or
D-Phe12
CRF12
41 antagonists (9, 21, 41,
47, 48). These findings would suggest that these stressors, unlike
abdominal surgery, selectively activate brain CRF pathways.
In summary, urocortin, the new mammalian member of the CRF family characterized as the endogenous ligand for CRF-R2, injected intravenously, dose dependently delays gastric emptying of a nonnutrient meal in conscious rats with a rank order of potency showing rat urocortin > rat CRF. The mixed CRF-R1/CRF-R2 antagonist astressin prevented CRF and urocortin inhibitory effects, whereas relatively large doses of the selective nonpeptide CRF-R1 antagonists antalarmine and NBI-27914 have no effect. In addition, we showed that peripheral injection of a low dose of astressin (12 µg/kg), which by itself did not influence basal gastric emptying, completely abolished gastric stasis observed 3 h after abdominal surgery while the CRF-R1 antagonists were inactive. These results suggest an important role of peripheral CRF and urocortin acting through CRF-R2 as part of the mechanisms involved in acute postoperative gastric ileus.
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ACKNOWLEDGEMENTS |
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We thank Dr. Georges P. Chrousos (Pediatric Endocrinology Section, National Institutes of Health, Bethesda, MD) for the generous supply of antalarmin and vehicle and Dr. Errol B. De Souza (Neurocrine Biosciences, San Diego, CA) for the generous supply of NBI-27914.
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FOOTNOTES |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33061 (Y. Taché), DK-41301 (Animal Core), and DK-26741 (J. Rivier).
Present address of V. Martinez: CEU-San Pablo, Veterinary School, Physiology Edificio Seminario, 46113, Moncada, Valancia, Spain (E-mail: vmartine{at}ceu.upv.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Taché, CURE: Digestive Diseases Research Center, West Los Angeles VA Medical Center, Bldg. 115, Rm. 203, 11301 Wilshire Blvd, Los Angeles, CA 90073 (E-mail: ytache{at}ucla.edu).
Received 11 September 1998; accepted in final form 5 December 1998.
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REFERENCES |
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M. Wald,
A. M. Bamberger,
S. Ergun,
F. U. Beil,
and
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Human lymphocytes produce urocortin, but not corticotropin-releasing hormone.
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Banks, W. A.,
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Passage of peptides across the blood-brain barrier: pathophysiological perspectives.
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