urocortin 2 acts centrally to delay gastric emptying through ... - CiteSeerX

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (October 13, 2005). doi:10.1152/ajpgi.00289.2005

UROCORTIN 2 ACTS CENTRALLY TO DELAY GASTRIC EMPTYING THROUGH SYMPATHETIC PATHWAYS WHILE CRF AND UROCORTIN 1 INHIBITORY ACTIONS ARE VAGAL DEPENDENT IN RATS

József Czimmer, Mulugeta Million, and Yvette Taché

CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences & Women’s Health, Department of Medicine, Division of Digestive Diseases, University of California Los Angeles, and VA Greater Los Angeles Healthcare System, Los Angeles, California 90073

Running head: CENTRAL UROCORTIN 2 INHIBITS GASTRIC EMPTYING

Contact information: Yvette Taché, Ph.D., CURE/CNS Bldg. 115, Room 117, VA Greater Los Angeles Healthcare System, 1130 Wilshire Blvd, Los Angeles, CA 90073, USA E-mail: [email protected]; Phone: (310) 312-9275; Fax (310) 268-4963

Copyright © 2005 by the American Physiological Society.

CENTRAL UROCORTIN 2 INHIBITS GASTRIC EMPTYING

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ABSTRACT We characterized the influence of the selective corticotropin-releasing factor 2 (CRF2) receptor agonist, urocortin 2 (Ucn 2), injected intracisternally (ic), on gastric emptying and its mechanism of action compared with ic CRF or urocortin (Ucn 1) in conscious rats. The methylcellulose phenol red solution was gavaged 20 min after peptide injection and gastric emptying measured 20 min later. The ic injection of Ucn 2 (0.1 and 1 µg) and Ucn 1 (1 µg) decreased gastric emptying to 37.8 ± 6.9%, 23.1 ± 8.6% and 21.6 ± 5.9%, respectively, compared with 58.4 ± 3.8% in ic vehicle. At lower doses, Ucn 2 (0.03 µg) and Ucn 1 (0.1 µg) had no effect. The CRF2 antagonist, astressin2-B (3 µg, ic), antagonized ic Ucn 2 (0.1 µg) and CRF (0.3 µg) -induced inhibition of gastric emptying. Vagotomy enhanced ic Ucn 2 (0.1 or 1 µg)-induced inhibition of gastric emptying compared to sham-operated group, while blocking ic CRF (1 µg) action (45.5 ± 8.4% vs. 9.7 ± 9.7%). Sympathetic blockade by bretylium prevented ic and intracerebroventricular Ucn 2-induced delayed gastric emptying, while not influencing intravenous Ucn 2-, ic CRF- and ic Ucn 1-induced inhibition of gastric emptying. Prazosin abolished ic Ucn 2 inhibitory effect, while yohimbine and propranolol did not. None of the pretreatment modified basal gastric emptying. These data indicate that ic Ucn 2 induced a central CRF2-mediated inhibition of gastric emptying involving sympathetic α1-adrenergic mechanisms independent from the vagus contrasting with the vagal-dependent inhibitory actions of CRF and Ucn 1.

Key words: Ucn 2, gastric emptying, CRF2, sympathetic, adrenergic receptors, vagus, rats


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INTRODUCTION Corticotropin-releasing factor (CRF) is a 41-amino acid peptide first isolated from ovine hypothalamus that plays a central role in the stress-related stimulation of the hypothalamic pituitary-adrenal (HPA) axis (2). Recently, three novel mammalian CRF-related peptides have been characterized (17). Rat and human urocortin 1(r/hUcn 1) are identical 40-amino acid peptides that share 43% identity with r/hCRF (17). The two novel putative urocortin isoforms, mouse urocortin 2 (mUcn 2) and mouse urocortin 3 (mUcn 3), are 38-amino acid peptides that are more distantly related to r/hCRF with 34% and 26% homology, respectively (17). The human Ucn 2 (hUcn 2) counterpart, also named urocortin-related peptide, shares 76% homology with mUcn 2 and is homologous to the postulated N-terminally extended 43-amino acid peptide stresscopin-related peptide (17). CRF ligands mediate their biological actions through interaction with two distinct receptors, subtype 1 (CRF1) and subtype 2 (CRF2), cloned from distinct genes (2). Both CRF1 and CRF2 receptors belong to the class B subfamily of seven-transmembrane receptors coupled to GS proteins (2). Radioreceptor and functional assays have demonstrated that CRF1 and CRF2 receptors differ considerably in their binding characteristics to natural CRF ligands (17). The CRF1 receptor shows no appreciable binding to Ucn 2 and Ucn 3, but binds with high affinity to Ucn 1, CRF and the amphibian CRF-related peptide, sauvagine (17). In contrast, the CRF2 receptor binds to Ucn 1, Ucn 2, Ucn 3 and sauvagine with a greater affinity than to CRF, making this receptor subtype highly selective for mammalian urocortin signaling (17). Activation of brain CRF receptors results in a number of stress-like responses (2) of which the inhibition of gastric transit under various experimental conditions has been reproducibly documented (42). In particular, initial reports showed that CRF injected


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intracerebroventricularly (icv) or intracisternally (ic) inhibits gastric emptying of a non-nutrient viscous methylcellulose solution in conscious female or male rats (43; 49). Other studies established that CRF injected into the cerebrospinal fluid at the level of the lateral or 4th ventricle or the cisterna magna inhibits gastric emptying of saline, glucose, acid, or a peptone liquid meal delivered intragastrically, as well as gastric emptying of a physiological meal (ingestion of solid chow) in conscious rats (reviewed in Taché, 1999 (42)). Likewise in other species, icv injection of CRF inhibits gastric emptying of a non-nutrient liquid and an ingested solid chow meal in mice (31; 40), and the total gastric emptying time of a solid nutrient meal in dogs (24). Consistent sets of observations established that the inhibition of gastric emptying induced by ic or icv injection of CRF in various species is not related to the activation of HPA axis and is mediated by the autonomic nervous system. Neither hypophysectomy nor acute adrenalectomy altered icv CRF-induced delayed gastric emptying in rats (6; 25; 43), while ganglionic blockade by chlorisondamine abolished icv CRF action in rats and mice (25; 40). Several reports showed that vagal-dependent mechanisms are involved in central CRF-induced delayed gastric emptying of a liquid non-caloric meal in rats and caloric meal in dogs (6; 24; 43). In contrast, two reports indicate the importance of sympathetic pathways in mediating ic or icv injection of CRF-induced delayed gastric emptying in rats (25; 34). Less is known on the central action of Ucns in inhibiting gastric motor function. A doserelated inhibition of gastric emptying of a solid meal has been reported after the ic injection of Ucn 1 in rats (10) and icv injection of Ucn 2 in mice (31). However, the pathways through which central injection of Ucn 1 and Ucn 2 inhibit gastric emptying, are still to be determined. Therefore, the aim of the present study was to characterize the central action of Ucn 2 on gastric emptying in conscious rats and to compare with that of CRF1/CRF2 agonists, Ucn 1 or CRF. We


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assessed the CRF2 receptor-mediated action of ic Ucn 2, Ucn 1 and CRF using the selective CRF2 antagonist, astressin2-B (38). Autonomic pathways through which ic and icv Ucn 2 and ic Ucn 1 inhibit gastric emptying were delineated using surgical (subdiaphragmatic vagotomy) and pharmacological (noradrenergic, α1-, α2-, and β-adrenergic receptor blockade) approaches.

MATERIALS AND METHODS

Animals Adult male Sprague-Dawley rats (Harlan, San Diego, CA) weighing 250-300 g were housed in group cages under controlled illumination (12:12 h light/dark-cycle starting at 6 AM), humidity and temperature (21-23°C), and had free access to tap water and Purina® rat chow. Rats were deprived of food, but had free access to tap water for 16-18 h before the experiments, except as otherwise stated. Protocols were approved by the UCLA and Veteran Affairs Greater Los Angeles Healthcare System Animal Research Committees (protocol no. 99127-07).

Compounds R/h CRF, r/h Ucn 1, hUcn 2, and astressin2-B (Peptide Biology Laboratory, The Salk Institute, La Jolla, CA) were synthesized using the solid phase approach and the Boc-strategy (38). Peptides were stored in powder form at –80°C. Immediately before the experiments CRF, Ucn 1 and Ucn 2 were dissolved in saline and astressin2-B in double-distilled water (pH 7.0). Bretylium tosylate, and propranolol hydrochloride (both from Sigma Chemical, St. Louis, MO) were stored at room temperature, and yohimbine hydrochloride (Sigma) and prazosin hydrochloride (Pfizer, Croton, CT) were stored at –20°C. Drugs were dissolved in sterile saline


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just before use, except yohimbine hydrochloride that was dissolved in 5% DMSO and 95 % distilled water. Phenol red and methylcellulose (both from Sigma) were stored at room temperature and prepared on the day of experiments. Ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (Rompun, Mobay Co., Shawnee, KS) were used to anesthetize rats undergoing surgery.

Treatments Intracisternal injections were performed as previously reported (43) under short isoflurane anesthesia (2-3 min, 5% vapor concentration in oxygen; VSS, Rockmart, GA). The occipital membrane was punctured with a 50 µl Hamilton syringe using stereotaxic equipment. Presence of cerebrospinal fluid into the Hamilton syringe upon aspiration before injection ensured the right needle tip position into the cisterna magna. The volume of injections was 10 µl for CRF, Ucn 1, and Ucn 2, and 5 µl for astressin2-B. Intracerebroventricular injections were performed as previously described (30) in rats equipped with a chronic icv cannula. Animals were anesthetized with an intraperitoneal (ip) injection of a mixture of ketamine hydrochloride (75 mg/kg) and xylazine (5 mg/kg). A guide cannula (22 ga, Plastic One Products, Roanoke, VA) was implanted into the right lateral brain ventricle according to the following coordinates (mm from bregma: antero-posterior, –0.8; lateral, –1.5; dorsoventral, –3.5). The guide cannula was maintained in place by dental cement anchored by four stainless steel jewelry screws fixed to the skull. After the surgery, animals were housed individually with direct bedding. Experiments were performed in conscious rats starting 10 days after the icv cannulation. During this time, rats were habituated to the manipulation of the cannula and handled daily for a period of about 5 min. The icv injection was performed in


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lightly restrainted rats and a 28 ga injection cannula, 1 mm longer than the guide cannula, was connected to a 50 µl Hamilton syringe by a PE-50 catheter (Intramedic Polyethylene Tubing, Clay Adams, Sparks, MD) filled with distilled water. A small air bubble (1 µl) was drawn at the distal end of the PE-50 catheter to separate the injected solution from the water and for visual inspection of the 10 µl injection which was performed slowly over 30 s. At the end of the experiments, the correct location of the cannula into the lateral ventricle was verified by injecting 10 µl of dye (0.1% toluidine blue). Visualization of dye on the wall of the lateral ventricle indicates correctness of the icv injections. Intravenous injections (0.1 ml) were performed into the right jugular vein after a skin incision under short (2-5 min) isoflurane anesthesia. Wounds were closed with sterile silk (2.0 metric, Ethicon Inc., Somerwille, NJ). Intraperitoneal (0.3 ml) and subcutaneous (0. 1 ml) injections were done in conscious rats. Subdiaphragmatic vagotomy was performed by circular seromuscular myotomy of the esophagus 2 cm proximal from the gastro-esophageal junction in fasted rats under ketamine hydrochloride (75 mg/kg, ip) and xylazine (25 mg/kg, ip) anesthesia. Sham vagotomy consisted of a laparotomy and similar manipulation of the esophagus and stomach without the myotomy under anesthesia. Rats that underwent subdiaphragmatic vagotomy or sham vagotomy were maintained with a liquid diet (Ensure; Ross Products Division, Abbott Laboratories, Columbus, OH) for 24 h post surgery, then deprived of liquid diet, but not water, for 12 h prior to the gastric emptying measurements .

Measurement of Gastric Emptying


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Gastric emptying of a non-nutrient viscous meal was determined by the phenol red method as described previously (43). The non-caloric meal consisted of a viscous suspension of continuously stirred 1.5% methylcellulose (w/v) containing phenol red (50 mg/100 ml) given intragastrically through a stainless steel gavage tube (in 1.5 ml volume) to conscious rats. At 20 min after the administration of the solution, rats were euthanized by CO2 inhalation. The abdominal cavity was opened, the gastro-esophageal junction and the pylorus were clamped, and the stomach was isolated and rinsed in 0.9% saline. The stomach was placed into 100 ml of 0.1 N NaOH and homogenized (Polytron; Brinkman Instruments, Inc., Westbury, NY). The suspension was allowed to settle for 60 min at room temperature, then 5 ml of supernatant was added to 0.5 ml of 20% trichloroacetic acid (w/v) and centrifuged at 3000 rpm at 4ºC for 20 min. After the supernatant was mixed with 4 ml of 0.5 N NaOH, the absorbance of the sample was read at 560 nm (Shimazu 260 Spectrophotometer). The absorbance of the phenol red recovered from animals euthanized immediately after gavage of the liquid meal was taken as a standard 0% emptying. The percentage of emptying during the 20-min period was calculated with the following formula: % emptying = (1 – absorbance of test sample/absorbance of standard) × 100.

Experimental Protocols Effect of ic Ucn 2, Ucn 1 and CRF alone or with CRF2 receptor antagonist on gastric emptying. Fasted rats were briefly anesthetized with isoflurane and injected ic with saline (10 µl), Ucn 2 (0.03, 0.1 or 1 µg), or Ucn 1 (0.1 or 1 µg). In another set of experiments, fasted rats were injected ic (5 µl) with double-distilled water or astressin2-B (1, 3 or 10 µg) just before ic injection of saline (10 µl), Ucn 2 (0.1 µg) or CRF (0.3 µg). At 20 min after ic injection, the


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phenol red methylcellulose solution was delivered intragastrically, and gastric emptying determined 20 min later. Effect of subdiaphragmatic vagotomy on ic injection of Ucn 2- and CRF-induced delayed gastric emptying. Sham or subdiaphragmatic vagotomy rats fasted for 12 h were injected ic with saline (10 µl), CRF (1 µg) or Ucn 2 (0.1 or 1 µg), and 20 min later animals received the gastric gavage of 1.5 ml phenol red methylcellulose solution. Gastric emptying was determined 20 min later. Effect of bretylium pretreatment on ic, icv and iv Ucn 2-, ic Ucn 1- and ic CRF-induced delayed gastric emptying. Bretylium tosylate (25 mg/kg) or saline was injected ip 60 min before Ucn 2 (0.1 µg ic, 1 µg icv or 15 µg/kg iv), Ucn 1 (1 µg, ic), CRF (0.3 µg, ic) or saline (ic, icv or iv). After 20 min, the 1.5 ml phenol red methylcellulose solution was given by gavage and gastric emptying monitored 20 min later. The dose of bretylium was based on a previous study showing complete noradrenergic blockade (25). The doses of Ucn 2 injected ic, icv and iv were based on dose response studies (present studies and data not shown) inducing a similar suppression of gastric emptying of a non-nutrient meal. Effect of α1-, α2-, and β-adrenergic blockade on ic Ucn 2-induced delayed gastric emptying. Prazosin (1 mg/kg), propranolol (1 mg/kg), or saline (0.3 ml) was injected ip 30 min before saline or Ucn 2 (0.1 µg, ic). In other studies, yohimbine or vehicle (5% DMSO/95% distilled water) was injected sc 30 min before ic injection of saline or Ucn 2 (0.1 µg). Twenty min later, rats were gavaged with 1.5 ml phenol red methylcellulose solution and euthanized after 20 min for gastric emptying determination. Doses of prazosin, yohimbine and propranolol were based on previous studies showing complete α1-, α2- and β-adrenergic receptor blockade, respectively, of peptide action on gut function (12; 29).


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Statistical Analysis All results are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test was performed for comparison between groups. P values < 0.05 were considered statistically significant.

RESULTS

Ucn 2 and Ucn 1 injected ic inhibit gastric emptying in conscious rats. Ucn 2, injected ic at 0.03, 0.1 and 1 µg, dose-dependently inhibits gastric emptying of a viscous non-caloric solution in conscious rats (Fig. 1). At 0.1 µg, ic Ucn 2 significantly reduced gastric emptying to 37.8 ± 6.9% compared with 58.4 ± 3.8% in the ic saline-treated group, while Ucn 1 had no effect (56.3 ± 0.5%). At 1 µg, both Ucn 2 and Ucn 1 injected ic induced a similar inhibition of gastric transit (23.1 ± 8.6% and 21.6 ± 5.9%, respectively, P < 0.05 compared with ic saline; Fig. 1). Astressin2-B injected ic prevents ic Ucn 2- and ic CRF-induced delayed gastric emptying. Astressin2-B, injected at 3 and 10 µg, completely prevented ic Ucn 2-induced inhibition of gastric emptying (51.6 ± 2.8% and 54.3 ± 7.2%, respectively, vs. 36.6 ± 6.2%, P < 0.05), while at 1 µg ic, astressin2-B was ineffective (27.9 ± 4.5%; Fig.2). Similarly, astressin2-B injected ic at 3 µg completely antagonized ic CRF (0.3 µg)-induced inhibition of gastric emptying (52.0 ± 4.6% vs. 18.9 ± 4.8% P < 0.05). Astressin2-B (3 µg) injected ic alone did not influence basal gastric emptying of a non-nutrient viscous meal (51.3 ± 6.6%; Fig. 2). Vagotomy blocked ic CRF, but not ic Ucn 2, -induced inhibition of gastric emptying. Ucn 2, injected ic at 0.1 µg, did not significantly delay gastric emptying in sham-vagotomized rats


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(29.0 ± 9.5% vs. 34.6 ± 3.8%, respectively), while there was a significant reduction in vagotomized rats (15.6 ± 3.2% vs. 33.4 ± 5.9%). At 1 µg, ic Ucn 2 significantly reduced gastric emptying in sham-group (16.9 ± 2.9% vs. 34.6 ± 3.8%), and the inhibitory effect was significantly enhanced in vagotomized (6.3 ± 3.0%) compared to sham (16.9 ± 2.9%) groups (Fig.3). In sham-vagotomized rats, ic injection of CRF (1 µg) significantly delayed gastric emptying (9.7 ± 9.7%) compared to vehicle (34.6 ± 3.8%). Subdiaphragmatic vagotomy completely prevented ic CRF-induced delayed gastric emptying (45.5 ± 8.4%; Fig. 3). Subdiaphragmatic vagotomy did not influence basal gastric emptying of viscous solution (33.4 ± 5.9%) compared with sham-operated rats (34.6 ± 3.8%; Fig. 3). It is of note that sham and vagotomized ic vehicle groups, fed a liquid diet for 24 h post surgery followed by a 12 h fast had lower gastric emptying compared with other non-operated ic vehicle groups (Fig. 3 vs. Fig. 1, 2-4,5) fasted for 16-18 h. The shorter fasting time period along with the surgery, which is known to influence gastric emptying (30; 48), may have contributed to the lower gastric emptying in operated rats. Bretylium prevents ic and icv Ucn 2-, but not iv Ucn 2-, ic CRF- or ic Ucn 1-induced inhibition of gastric emptying. Ucn 2, injected ic (0.1 µg) in rats under short anesthesia or icv (1 µg) in lightly restrained conscious rats, significantly delayed gastric emptying (27.1 ± 7.3% and 22.7 ± 5.7%, respectively) compared to groups injected with saline either ic (55.8 ± 6.8%) or icv (67.6 ± 13.5%; Fig. 4). The inhibitory effect of both ic and icv Ucn 2 was completely prevented by ip bretylium tosylate (56.1 ± 4.4% and 57.0 ± 8.7%, respectively; Fig. 4). In contrast, neither ic CRF (0.3 µg) nor ic Ucn 1 (1 µg) -induced delayed gastric emptying (25.3 ± 4.0% and 19.7 ± 5.7, respectively) was altered by pretreatment with ip bretylium tosylate (18.5 ± 4.9% and 11.9 ± 4.4%, respectively; Fig. 4). In addition, iv Ucn 2-induced significant suppression of gastric


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emptying (9.0 ± 3.2%) was not modified by bretylium tosylate pretreatment (5.0 ± 3.5%; Fig. 4C). In ic, icv or ip saline treated groups, ip bretylium tosylate did not modify gastric emptying compared to ip vehicle (Fig. 4A-C). Prazosin prevents ic Ucn 2-induced inhibition of gastric emptying, while yohimbine and propranolol had no effect. Pretreatment with the α1-adrenergic receptor blocker, prazosin (1 mg/kg, ip), completely abolished the delayed gastric emptying induced by ic Ucn 2 compared with vehicle pretreated group (56.9 ± 9.3% vs. 29.2 ± 3.0%). In contrast the α2-adrenergic receptor blocker, yohimbine (4 mg/kg, sc), and β-adrenergic receptor blocker, propranolol (1 mg/kg, ip), did not modify Ucn 2 (0.1 µg, ic)-induced delayed gastric emptying (Fig.5; Table 1). Prazosin, yohimbine and propranolol injected alone with ic saline did not significantly alter the 20 min basal gastric emptying values (Fig. 5; Table 1).

DISCUSSION

The present study demonstrates that the newly characterized CRF2 selective agonist, Ucn 2 injected ic, inhibits gastric emptying of a viscous non-caloric solution in a dose-dependent manner in conscious fasted rats. Ucn 2 injected ic is more potent than Ucn 1 as shown by the significant inhibition of gastric emptying induced by Ucn 2 at 0.1 µg (24 pmol), while Ucn 1 (0.1 µg, 21 pmol) had no effect under these conditions. However, at 1 µg both Ucn 1 and Ucn 2 exert a similar 60% and 63% reduction of gastric emptying, respectively. These observations expend previous reports showing that Ucn 1, injected ic at 0.3 µg (63 pmol), did not alter gastric emptying of a solid meal, while inducing a dose-related inhibition at ic doses of 0.6 and 1 µg (10). Likewise, CRF injected ic at 0.1 µg (21 pmol) had no effect on gastric emptying of a


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methylcellulose solution while significantly suppressing gastric emptying when injected ic at 0.3-1 µg in conscious rats (28; 43) (present study). Although gastric emptying was monitored in fasted rats briefly anesthetized with isoflurane for the duration of the puncture of the cisterna magna and ic injection, it is unlikely that the peptide inhibitory action represents an interaction with the anesthesia or other stressful aspects of peptide injection. We observed a similar delay in gastric emptying in response to Ucn 2 injected in conscious fasted rats through a chronically implanted icv cannula (present study) or in fed or fasted conscious rats injected icv with CRF (25; 28). The selective CRF2 antagonist, astressin2-B (38) injected ic, completely prevented both ic Ucn 2- and ic CRF-induced delayed gastric emptying of a liquid non-nutrient meal (present study) and ic Ucn 1-induced delayed gastric emptying of a chow meal (10). The delayed gastric emptying induced by ic injection of the selective CRF2 agonist, Ucn 2, and the prevention of ic Ucn 2, ic CRF and ic Ucn 1 inhibitory action by the selective CRF2 antagonist provide direct pharmacological evidence that the CRF2 receptor is involved in ic CRF and urocortin action. Before the identification of CRF2 agonists and antagonists, indirect evidence were indicative of a role of brain CRF2 receptors based on the rank order of potency of ic sauvagine>urotensinI>CRF to inhibit gastric emptying of a solid meal in rats (28), which was in line with their differential affinity to CRF2 receptors (sauvagine>urotensin-I>CRF) (35). Convergent evidence indicates that sympathetic pathways and α1-adrenergic receptors are involved in the inhibition of gastric emptying induced by central injection of Ucn 2. First, ip injection of bretylium, an adrenergic neuronal blocking agent taken up selectively at peripheral adrenergic nerve terminals, and blocking transmitter release from sympathetic postganglionic nerve terminals (7) abolished both ic and icv Ucn 2-induced decreased gastric emptying. Second,


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subdiaphragmatic vagotomy did not block, but significantly enhanced, the inhibitory action of Ucn 2 injected ic at 0.1 and 1 µg resulting in a 56% and 82% suppression of gastric emptying. The increased inhibitory effect of ic Ucn 2 in vagotomized rats may be consistent with the sympathetic mediated inhibitory mechanisms that are no longer restrained by vagal cholinergic tone (1). The main neurotransmitters/neuromodulators in postganglionic sympathetic nerves are noradrenaline, ATP and neuropeptide Y (8). In the present study, pharmacological blockade of α1-adrenergic receptors by prazosin mimicked the effects of bretylium while α2- and β-adrenergic blockade by yohimbine and propranolol had no effect. Such a reversal of ic Ucn 2 action occurs under conditions where sympathetic or adrenergic blockade did not alter basal gastric emptying as previously reported for gastric emptying and motility in fasted and fed state rats (22; 25). In rats, norepinephrine injected iv induces fundic relaxation (3) and the α1-agonist L-phenylephrine reduced the amplitude of gastric phasic contractions while β-agonists, prenalterol or salbutamol, did not (4). Gastric relaxation and α1 receptor-mediated inhibition of phasic gastric contraction may play a role in the sympathetic α-adrenergic mediated delayed gastric emptying induced by ic Ucn 2. Ucn 2 injected iv significantly suppressed gastric emptying of a liquid non-nutrient meal (present study) and of a solid meal in conscious rats (33). However, the inhibitory action of Ucn 2 injected iv was not altered by bretylium pretreatment under conditions blocking ic or icv Ucn 2-induced delayed gastric emptying. These results established that the systemic inhibitory action of Ucn 2 is mediated through distinct mechanisms from those elicited by central administration. In addition, these data indicate that ic or icv Ucn 2 did not act by leaking into the periphery but is centrally mediated through stimulation of sympathetic pathways. In this regard, since prazosin after its peripheral injection can penetrate the blood-brain barrier and decrease the sympathetic


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nerve activity in rats (39), it is possible that prazosin in the present study also acts in the brain to block the sympathetic mediated action of ic Ucn 2. So far, two reports indicate that the activation of brain CRF2 receptors influences other visceral functions through sympathetic pathways (11; 50). The selective CRF2 agonist, urocortin 3 (17), increases mean arterial blood pressure, heart rate and plasma epinephrine release in conscious rats consistent with activation of sympathetic outflow (11). The ic injection of Ucn 2 was also recently shown to decrease hepatic surface perfusion and elevate portal pressure through CRF2 receptors and sympathetic noradrenergic mechanisms in anesthetized rats (50). Interestingly, consistent reports indicate that Ucn 2, injected icv at 1 µg, displays satiation-like properties that occurred 3-6 h after peptide injection in conscious rats (20). In our study, the inhibition of gastric emptying induced by icv Ucn 2 at 1 µg was observed within the first hour. Whether gastric fullness associated with altered gastric emptying underlies the delayed inhibition of feeding behavior in response to icv Ucn 2 may need to be considered. Previous observations indicate that gastric distention and the presence of food in the stomach act as satiety signals (36). The exact brain sites at which ic or icv Ucn 2 induces a centrally mediated sympathetic inhibition of gastric motor function are still to be established. In the medulla oblongata, specific CRF2 binding sites detected by autoradiography are restricted to the nucleus tractus solitarius (NTS) and area postrema, and mRNA encoding the CRF2 receptor is also densely expressed in both of these structures (27; 45). In addition, icv injection of hUcn 2 at 1 µg in conscious rats gives rise to Fos expression, indicative of neuronal activation in the NTS, the paraventricular nucleus of the hypothalamus (PVN) and central amygdala, while no Fos induction was observed in the locus coeruleus, dorsal motor nucleus (DMN) or area postrema (20; 37). Based on these neuroanatomical and neurofunctional studies, it is likely that ic and icv Ucn 2 act at these


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responsive brain sites to influence sympathetic outflow and inhibit gastric motor function (13; 15). The present studies also provide evidence that ic CRF and Ucn 1 inhibit gastric emptying of a non-nutrient meal through distinct neural pathways than Ucn 2. Bretylium injected under conditions that completely antagonized ic or icv Ucn 2 action, did not alter either CRF or Ucn 1 injected ic at doses resulting in a similar percentage of gastric emptying as ic Ucn 2. Moreover, subdiaphragmatic vagotomy prevented ic CRF-induced inhibition of gastric emptying, while not blocking that of ic Ucn 2 action. Subdiaphragmatic vagotomy did not significantly alter the transit of a liquid caloric meal compared with sham surgery in ic vehicle injected rats as previously reported (25; 43). Consistent with the vagal pathway involved in ic CRF and Ucn 1 inhibitory action, previous studies showed that subdiaphragmatic vagotomy blocked ic or icv CRF-induced delayed gastric emptying of a liquid non-nutrient meal in rats (6; 43). Vagotomy also blocked gastric motility changes induced by CRF injected into the DMN in fasted rats or icv in fed dogs (24; 26), and Ucn 1 injected icv, while surgical sympathectomy had no effect in fed or fasted rats (22). In addition, CRF injected ic or into the DMN inhibits gastric motility stimulated by vagal-dependent TRH excitatory action on DMN neurons (10; 19). In support of vagal inhibitory pathways are also the observations that ic CRF decreased gastric vagal efferent discharge (23) and icv CRF blocked the activation of DMN neurons and gastric function induced by endogenous TRH released by acute cold exposure (47). In contrast, an in vitro patch clamp study performed on coronal sections containing the dorsal vagal complex of 25 day old rats showed that superfusion of CRF increases discharge rate and membrane depolarization of gastric projecting DMN neurons (26). Age difference and in vitro vs. in vivo experimental conditions recruiting different circuitries could account for these divergent results. Interestingly there are


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two reports of a sympathetic mediated central action of CRF to suppress gastric emptying when injected ic before the ingestion of a solid meal (34) or icv before a liquid acaloric meal in conscious rats (25). Whether vagal vs. sympathetic actions of CRF are related to different experimental conditions or dose-related effects mimicking the sympathetic mediated action of Ucn 2 under these conditions needs to be further ascertained. Although CRF displays high affinity to CRF1 and a 40-fold lower affinity to CRF2 receptors, and Ucn 1 has a high affinity to both CRF receptor subtypes (17), the selective CRF2 antagonist, astressin2-B, antagonized ic CRF- and Ucn 2-induced inhibition of gastric emptying of a liquid meal (present study) and ic Ucn 1-induced inhibition of a solid nutrient meal (10). We previously established the biological CRF2 selectivity of astressin2-B in rats by showing that the peptide did not influence the established CRF1 receptor-mediated action of iv CRF on the stimulation of colonic motor function under conditions antagonizing the CRF2-mediated effect of iv CRF on the stomach (33). These data support that ic CRF and Ucn 1 interact with CRF2 receptors to induce a vagally-mediated inhibition of gastric motor function. However, it cannot be discounted that the astressin2-B-sensitive effect of CRF/Ucn 1 may reflect coactivation of CRF1 and CRF2 receptors or initial activation of CRF1 pathways that recruit brain medullary CRF2 receptors. The underlying mechanisms whereby preferential vagal vs. sympathetic pathways are recruited through activation of brain CRF2 receptors by different members of the CRF family are still to be defined. Ucn 2 and CRF/Ucn 1 display differential chemical properties that may play a role (5). hUcn 2 binds with high and selective affinity to CRF2 receptors, low affinity to CRF binding protein, and lacks affinity to the recently cloned soluble splice variant, sCRF2α, identified in rat and mice brain, including the pons/medulla (9; 18; 21). In contrast, Ucn 1 and


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CRF bind to both CRF receptor subtypes and show subnamolar affinity to the CRF binding protein and sCRF2α (9; 21; 46), which may decrease their availability to CRF2 receptors located at more distant sites of injection. For instance, recent studies showed that Ucn 2 injected icv activates serotonergic neurons in the dorsal region of the mid-rostrocaudal and caudal subdivision of the dorsal raphe nucleus in close proximity of the aqueduct (41), where CRF2 receptors are abundantly expressed (16). Ucn 2 injected icv and ic may have a wide spread distribution on dorsal raphe neurons and induce sympathetic-mediated inhibition of gastric motor function as demonstrated for the sympathetic-mediated vascular response to activation of similar regions of dorsal raphe neurons (32). The possibility of a third CRF receptor that is recognized by astressin2-B and mediates the differential mechanisms of action of CRF/Ucn 1 and Ucn 2 cannot be ruled out. The notion of additional CRF receptor subtypes has been brought forward previously on the basis of distinct biological responses to CRF linked with differential antagonist:agonist ratios (14). In the present study, the antagonist:agonist ratio of 30:1 was required for astressin2-B to block ic Ucn 2 action, while a 10:1 ratio was ineffective. In contrast, at a 10:1 ratio, astressin2-B completely antagonized the inhibitory effect of ic CRF (present study) and ic Ucn 1 (10). Brain Ucn 2 may play a role in stress-related alterations of gastric motor function. First, we observed a potent action of ic Ucn 2 to suppress gastric emptying. Second, there is a similar central CRF2 receptor-mediated sympathetic inhibition of gastric emptying induced by icv or ic Ucn 2 (present study) and restraint stress (25; 44). Lastly, recent reports showed the presence of Ucn 2 mRNA in stress-responsive hypothalamic and brainstem nuclei (37), along with dramatic induction of Ucn 2 mRNA in the parvocellular part of the PVN by restraint (44).


19

In summary, the present study established that ic or icv injection of the selective CRF2 agonist, Ucn 2, acts centrally on CRF2 receptors to inhibit gastric emptying of a liquid meal through sympathetic pathways and α1-adrenoreceptor-dependent mechanisms. This was shown by the blockade of the gastric inhibitory response by ic injection of the selective CRF2 antagonist, astressin2-B, and peripheral injection of adrenergic blockers, bretylium and α1 antagonist, prazosin, while vagotomy, propranolol or yohimbine did not prevent ic Ucn 2 action. CRF and Ucn 1, also inhibit gastric emptying through activation of CRF2 receptors, however, their action is vagal-dependent and not altered by bretylium. Ucn 2 action is more potent than Ucn 1 and requires a 3-fold higher antagonist:agonist ratio. These data provide evidence for a differential sympathetic α1 adrenergic pathway recruited by the selective CRF2 agonist, Ucn 2 injected icv or ic, compared with vagal-dependent mechanisms involved in the ic CRF1/CRF2 agonists, CRF and Ucn 1, to inhibit gastric emptying of a liquid non-caloric meal.

CENTRAL UROCORTIN 2 INHIBITS GASTRIC EMPTYING ACKNOWLEDGEMENTS This work was supported by NIH grants, R01 DK-33061, Center grant DK-41301 (Animal Core), and VA Career Scientist and Merit Award. We thank Dr. Jean Rivier (Salk Institute, La Jolla, CA) for the generous supply of peptides, and Miss Teresa Olivas for help in the preparation of the manuscript. Dr. Jozsef Czimmer was supported partly by the B-142 Ph.D. program of the Faculty of Medicine of the University of Pecs, Hungary.

20


21

REFERENCES 1. Abrahamsson H and Glise H. Sympathetic nervous control of gastric motility and interaction with vagal activity. Scand J Gastroenterol Suppl 89: 83-87, 1984. 2. Bale TL and Vale WW. CRF and CRF receptor: Role in stress responsivity and other behaviors. Annu Rev Pharmacol Toxicol 44: 525-557, 2004. 3. Boeckxstaens GE, Hirsch DP, Kodde A, Moojen TM, Blackshaw A, Tytgat GN and Blommaart PJ. Activation of an adrenergic and vagally-mediated NANC pathway in surgery-induced fundic relaxation in the rat. Neurogastroenterol Motil 11: 467-474, 1999. 4. Bojo L, Cassuto J, Nellgard P and Jonsson A. Adrenergic, cholinergic and VIP-ergic influence on gastric phasic motility in the rat. Acta Physiol Scand 150: 67-73, 1994. 5. Brauns O, Liepold T, Radulovic J and Spiess J. Pharmacological and chemical properties of astressin, antisauvagine-30 and alpha-helCRF: significance for behavioral experiments. Neuropharmacology 41: 507-516, 2001. 6. Broccardo M and Improta G. Pituitary-adrenal and vagus modulation of sauvagine- and CRF-induced inhibition of gastric emptying in rats. Eur J Pharmacol 182: 357-362, 1990. 7. Brock JA and Cunnane TC. Studies on the mode of action of bretylium and guanethidine in post-ganglionic sympathetic nerve fibres. Naunyn Schmiedeberg's Arch Pharmacol 338: 504-509, 1988. 8. Burnstock G. Structural and chemical organization of the autonomic neuroeffector system. edited by Bolis CL., Licinio J and Govoni S. New York: Marcel Dekker Inc., 2004, p. 1-53. 9. Chen AM, Perrin MH, Digruccio MR, Vaughan JM, Brar BK, Arias CM, Lewis KA, Rivier JE, Sawchenko PE and Vale WW. A soluble mouse brain splice variant of type


22

2{alpha} corticotropin-releasing factor (CRF) receptor binds ligands and modulates their activity. Proc Natl Acad Sci U S A 102: 2620-2625, 2005. 10. Chen CY, Million M, Adelson DW, Martinez V, Rivier J and Taché Y. Intracisternal urocortin inhibits vagally stimulated gastric motility in rats: role of CRF(2). Br J Pharmacol 136: 237-247, 2002. 11. Chu CP, Qiu DL, Kato K, Kunitake T, Watanabe S, Yu NS, Nakazato M and Kannan H. Central stresscopin modulates cardiovascular function through the adrenal medulla in conscious rats. Regul Pept 119: 53-59, 2004. 12. Cooper SM and McRitchie B. Role of dopamine and a-adrenoreceptors in the control of gastric emptying in the rat: possible involvement in the mechanism of action of metoclopramide. J Auton Pharmacol 5: 325-331, 1985. 13. Coote JH, Yang Z, Pyner S and Deering J. Control of sympathetic outflows by the hypothalamic paraventricular nucleus. Clin Exp Pharmacol Physiol 25: 461-463, 1998. 14. Fisher L, Rivier C, Rivier J and Brown M. Differential antagonist activity of α-helical CRF9-41 in three bioassay systems. Endocrinology 129: 1312-1316, 1991. 15. Gillis RA, Quest JA, Pagani FD and Norman WP. Control centers in the central nervous system for regulating gastrointestinal motility. In: Handbook of Physiology-The Gastrointestinal System, Vol. 1 Motility and Circulation, edited by Wood JD and Schultz SC. New York: Oxford University Press, 1989, p. 621-683. 16. Hammack SE, Schmid MJ, LoPresti ML, Der-Avakian A, Pellymounter MA, Foster AC, Watkins LR and Maier SF. Corticotropin releasing hormone type 2 receptors in the dorsal raphe nucleus mediate the behavioral consequences of uncontrollable stress. J Neurosci 23: 1019-1025, 2003.


23

17. Hauger RL, Grigoriadis DE, Dallman MF, Plotsky PM, Vale WW and Dautzenberg FM. International Union of Pharmacology. XXXVI. Current Status of the Nomenclature for Receptors for Corticotropin-Releasing Factor and Their Ligands. Pharmacol Rev 55: 21-26, 2003. 18. Henry BA, Lightman SL and Lowry CA. Distribution of corticotropin-releasing factor binding protein-immunoreactivity in the rat hypothalamus: association with corticotropinreleasing factor-, urocortin 1- and vimentin-immunoreactive fibres. J Neuroendocrinol 17: 135-144, 2005. 19. Heymann-Monnikes I, Taché Y, Trauner M, Weiner H and Garrick T. CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acidstimulated gastric contractility in rats. Brain Res 554: 139-144, 1991. 20. Inoue K, Valdez GR, Reyes TM, Reinhardt LE, Tabarin A, Rivier J, Vale WW, Sawchenko PE, Koob GF and Zorrilla EP. Human urocortin II, a selective agonist for the type 2 corticotropin-releasing factor receptor, decreases feeding and drinking in the Rat. J Pharmacol Exp Ther 305: 385-393, 2003. 21. Jahn O, Tezval H, van Werven L, Eckart K and Spiess J. Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity. Neuropharmacology 47: 233-242, 2004. 22. Kihara N, Fujimura M, Yamamoto I, Itoh E, Inui A and Fujimiya M. Effects of central and peripheral urocortin on fed and fasted gastroduodenal motor activity in conscious rats. Am J Physiol 280: G406-G419, 2001.


24

23. Kosoyan HP, Wei JY and Taché Y . Intracisternal sauvagine is more potent than corticotropin-releasing factor to decrease gastric vagal efferent activity in rats. Peptides 20: 851-858, 1999. 24. Lee C and Sarna SK. Central regulation of gastric emptying of solid nutrient meals by corticotropin releasing factor. Neurogastroenterol Mot 9: 221-229, 1997. 25. Lenz HJ, Burlage M, Raedler A and Greten H. Central nervous system effects of corticotropin-releasing factor on gastrointestinal transit in the rat. Gastroenterology 94: 598-602, 1988. 26. Lewis MW, Hermann GE, Rogers RC and Travagli RA. In vitro and in vivo analysis of the effects of corticotropin releasing factor on rat dorsal vagal complex. J Physiol 543: 135146, 2002. 27. Li C, Vaughan J, Sawchenko PE and Vale WW. Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression. J Neurosci 22: 991-1001, 2002. 28. Martinez V, Barquist E, Rivier J and Taché Y. Central CRF inhibits gastric emptying of a nutrient solid meal in rats: the role of CRF2 receptors. Am J Physiol 274: G965-G970, 1998. 29. Martinez V, Cuttitta F and Taché Y. Central action of adrenomedullin to inhibit gastric emptying in rats. Endocrinology 138: 3749-3755, 1997. 30. Martinez V, Rivier J, Wang L and Taché Y. Central injection of a new corticotropinreleasing factor (CRF) antagonist, astressin, blocks CRF- and stress-related alterations of gastric and colonic motor function. J Pharmacol Exp Ther 280: 754-760, 1997.


25

31. Martinez V, Wang L, Rivier J, Grigoriadis D and Taché Y. Central CRF, urocortins and stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors delays gastric transit in mice. J Physiol 556.1: 221-234, 2004. 32. Mattila J and Bunag RD. Pressor and sympathetic responses to dorsal raphe nucleus infusions of TRH in rats. Am J Physiol 258: R1464-R1471, 1990. 33. Million M, Maillot C, Saunders PR, Rivier J, Vale W and Taché Y. Human urocortin II, a new CRF-related peptide, displays selective CRF2-mediated action on gastric transit in rats. Am J Physiol 282: G34-G40, 2002. 34. Nakade Y, Tsuchida D, Fukuda H, Iwa M, Pappas TN and Takahashi T. Restraint stress delays solid gastric emptying via a central CRF and peripheral sympathetic neuron in rats. Am J Physiol Regul Integr Comp Physiol 288: R427-R432, 2005. 35. Perrin MH and Vale WW. Corticotropin releasing factor receptors and their ligand family. Ann N Y Acad Sci 885: 312-328, 1999. 36. Phillips RJ and Powley TL. Gastric volume rather than nutrient content inhibits food intake. Am J Physiol 271: R766-R779, 1996. 37. Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, Hogenesch JB, Gulyas J, Rivier J, Vale WW and Sawchenko PE. Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci U S A 98: 2843-2848, 2001. 38. Rivier J, Gulyas J, Kirby D, Low W, Perrin MH, Kunitake K, DiGruccio M, Vaughan J, Reubi JC, Waser B, Koerber SC, Martinez V, Wang L, Taché Y and Vale W. Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists. J Med Chem 45: 4737-4747, 2002.


26

39. Saito H, Togashi H and Yoshioka M. A comparative study of the effects of alpha 1adrenoceptor antagonists on sympathetic function in rats. Am J Hypertens 9: 160S-169S, 1996. 40. Sheldon RJ, Qi JA, Porreca F and Fisher LA. Gastrointestinal motor effects of corticotropin-releasing factor in mice. Regul Pept 28: 137-151, 1990. 41. Staub DR, Spiga F and Lowry CA. Urocortin 2 increases c-Fos expression in topographically organized subpopulations of serotonergic neurons in the rat dorsal raphe nucleus. Brain Res 1044: 176-189, 2005. 42. Taché Y. Cyclic vomiting syndrome: the corticotropin-releasing-factor hypothesis. Dig Dis Sci 44: 79S-86S, 1999. 43. Taché Y, Maeda-Hagiwara M and Turkelson CM. Central nervous system action of corticotropin-releasing factor to inhibit gastric emptying in rats. Am J Physiol 253: G241G245, 1987. 44. Tanaka Y, Makino S, Noguchi T, Tamura K, Kaneda T and Hashimoto K. Effect of stress and adrenalectomy on urocortin II mRNA expression in the hypothalamic paraventricular nucleus of the rat. Neuroendocrinology 78: 1-11, 2003. 45. Van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C, Prins GS, Perrin M, Vale W and Sawchenko PE. Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. J Comp Neurol 428: 191-212, 2000. 46. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Rivier J, Sawchenko PE and Vale W. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378: 287-292, 1995.


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47. Wang L, Cardin S, Martinez V and Taché Y. Intracerebroventricular CRF inhibits cold restraint-induced c-fos expression in the dorsal motor nucleus of the vagus and gastric erosions in rats. Brain Res 736: 44-53, 1996. 48. Wang L, Martinez V, Rivier JE and Taché Y. Peripheral urocortin inhibits gastric emptying and food intake in mice: differential role of CRF receptor 2. Am J Physiol Regul Integr Comp Physiol 281: R1401-R1410, 2001. 49. Williams CL, Peterson JM, Villar RG and Burks TF. Corticotropin-releasing factor directly mediates colonic responses to stress. Am J Physiol 253: G582-G586, 1987. 50. Yoneda M, Nakamura K, Nakade Y, Tamano M, Kono T, Watanobe H, Shimada T, Hiraishi H and Terano A. Effect of central corticotropin releasing factor on hepatic circulation in rats: the role of the CRF2 receptor in the brain. Gut 54: 282-288, 2005.


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Table 1. Influence of yohimbine on ic Ucn 2-induced delayed gastric emptying in rats. Treatmenta

N

Gastric emptying (%)b

Vehicle + saline

5

58.9 ± 7.9

Yohimbine + saline

4

44.6 ± 12.6

Vehicle + Ucn2

7

30.5 ± 6.0*

Yohimbine + Ucn2

5

35.6 ± 4.2*

a

Rats fasted for 16-18 h were injected ic under short isoflurane anesthesia with saline or Ucn 2

(0.1 µg 30 min after vehicle or yohimbine (4 mg/kg). Twenty minutes after the ic injection, all groups were given an orogastric acaloric liquid meal and 20 min later gastric emptying was measured. b

Each value is the mean ± SEM of number of rats. * P < 0.05 compared with vehicle + saline.


29

FIGURE LEGENDS Fig. 1. Human urocortin 2 (Ucn 2) and rat urocortin (Ucn 1) injected intracisternally (ic) decreased gastric emptying of a viscous non-caloric meal in conscious rats. Rats under short anesthesia were injected ic with saline or peptide, and 20 min later, awake rats were gavaged with 1.5 ml of a non methylcellulose phenol red solution. Gastric emptying was monitored 20 min later. Each column is mean ± SEM of number of rats indicated in parenthesis. *P < 0.05 compared to ic saline group.

Fig. 2. The selective CRF2 antagonist, astressin2-B, injected ic prevents ic Ucn 2- and ic CRFinduced delayed gastric emptying of a viscous non-caloric meal in conscious rats. Astressin2-B or vehicle was injected ic before ic injection of saline, Ucn 2 or CRF. The protocol was as detailed in Fig. 1. Each column is the mean ± SEM of number of rats indicated in parenthesis. *P < 0.05 compared with ic vehicle + saline control group, and # compared to ic vehicle + Ucn 2 or CRF, respectively.

Fig. 3. Subdiaphragmatic vagotomy prevents ic CRF, but not Ucn 2, -induced delayed gastric emptying of a viscous non-caloric meal in conscious rats. Sham operation or vagotomy was performed 48 h before the experiments and 12 h fasted rats were injected ic with saline, CRF or Ucn 2. Gastric emptying was monitored as detailed in Fig. 1. Each column represents the means ± SEM of number of rats indicated in parenthesis. *P < 0.05 compared to sham operated + ic saline control group, and # compared with respective sham + ic CRF or Ucn 2 group.


30

Fig. 4. Bretylium tosylate prevented ic and icv, but not iv, injected Ucn 2-induced delayed gastric emptying of a viscous non-caloric meal in conscious rats. Fasted rats were injected ip with saline or bretylium 60 min before the ic (A) or iv (C) injection of peptides under light anesthesia or icv in conscious rats with chronic icv cannula (B). Gastric emptying was monitored as detailed previously. Each bar is the mean ± SEM of number of rats indicated in parenthesis. *P < 0.05 compared to saline ip + ic saline control group, and # compared with respective ip saline + ic, icv or iv Ucn 2 groups.

Fig. 5. Prazosin, unlike propanolol, prevents ic Ucn 2-induced delayed gastric emptying of a viscous non-caloric meal in conscious rats. The ip injection of propanolol or saline was performed 30 min, and that of prazosin or saline 30 min, before the ic injection of saline or Ucn 2, and gastric emptying was monitored 20 min later as detailed in the legend of Fig. 1. Each bar is the mean ± SEM of number of rats indicated in parenthesis. *P < 0.05 compared to saline ip + ic saline control group, and # compared with respective ip saline + ic Ucn 2 groups.


31

Figure 1

Gastric Emptying (% in 20 min)

100 Saline Ucn 2 Ucn 1

80

60

* 40

*

*

20

0

(8)

(4)

(7)

(4)

(5)

(4)

0

0.03

0.1

1

0.1

1

Peptide Dose (µg, ic)


32

Figure 2


100

Saline (10 µl, ic) Ucn 2 (0.1 µg, ic) CRF (0.3 µg, ic)

80

# 60

*

40

*

*

20

0

#

#

(10)

(4)

(8)

(4)

(4)

(4)

(4)

(6)

0

3

0

1

3

10

0

3

Astressin2-B (µg/rat, ic)


33

Figure 3


100

80

Sham Vagotomy

Saline (10 µl, ic)

Ucn 2

CRF

60

#

40

*

20

0

*

* *

(6)

(5)

(4)

(4)

(4)

(4)

(5)

(4)

0

0

1

1

0.1

0.1

1

1

Peptide Dose (µg, ic)


A Gastric Emptying (% in 20 min)

100

Saline (0.3 ml, ip) Bretylium tosylate (25 mg/kg, ip)

80

CRF (ic)

B

Ucn 1 (ic)

Ucn 2 (ic)

#

60

40

*

20

0

*

*

* *

(6)

(5)

(4)

(7)

(6)

(8)

(4)

(5)

0

0

0.3

0.3

1

1

0.1

0.1 (µg, ic)


100

80

** 60

40

C

*

20

0

Saline (icv)

Ucn 2 (1 µg, icv)

100


Figure 4

34

80

60

40

20

0

* Saline (iv)

*

Ucn 2 (15 µg/kg, iv)


35

Figure 5 Saline (10 µl, ic) Propranolol (1 mg/kg, ip) Prazosin (1 mg/kg, ip)


100

80

# 60

40

*

*

20

0

(7)

(7)

(5)

(5)

(5)

(5)

0

0

0

0.1

0.1

0.1

Ucn 2 (µg, ic)